β-sheet mimetics and use thereof as protease inhibitors

ABSTRACT

There are disclosed β-sheet mimetics and methods relating to the same for imparting or stabilizing the β-sheet structure of a peptide, protein or molecule. In one aspect, the β-sheet mimetics are covalently attached at the end or within the length of the peptide or protein. The β-sheet mimetics have utility as protease inhibitors generally, including activity as serine protease inhibitors such as thrombin, elastase and Factor X.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.09/009,386, filed Jan. 20, 1998, now U.S. Pat. No. 6,020,331 which is acontinuation of U.S. patent application Ser. No. 08/624,695, filed Mar.25, 1996, now abandoned; which is a continuation-in-part of U.S. patentapplication Ser. No. 08/549,006, filed Oct. 27, 1995, now abandoned;which is a continuation-in-part of U.S. patent application Ser. No.08/410,518, filed Mar. 24, 1995, now abandoned.

TECHNICAL FIELD

This invention relates generally to β-sheet mimetics and, morespecifically, to β-sheet mimetics for use as protease inhibitors.

BACKGROUND OF THE INVENTION

The β-sheet conformation (also referred to as a β-strand conformation)is a secondary structure present in many polypeptides. The β-sheetconformation is nearly fully extended, with axial distances betweenadjacent amino acids of approximately 3.5 Å. The β-sheet is stabilizedby hydrogen bonds between NH and CO groups in different polypeptidesstrands. Additionally, the dipoles of the peptide bonds alternate amongthe strands which imparts intrinsic stability to the β-sheet. Theadjacent strands in the β-sheet can run in the same direction (i.e., aparallel β-sheet) or in opposite directions (i.e., an antiparallelβ-sheet). Although the two forms differ slightly in dihedral angles,both are sterically favorable. The extended conformation of the β-sheetconformation results in the amino acid side chains protruding onalternating faces of the β-sheet.

The importance of β-sheets in peptides and proteins is well established(e.g., Richardson, Nature 268:495-499, 1977; Halverson et al., J. Am.Chem Soc. 113:6701-6704, 1991; Zhang, J. Biol. Chem. 266:15591-15596,1991; Madden et al., Nature 353:321-325, 1991). The β-sheet is importantin a number of biological recognition events, including the interactionbetween proteases and proteolytic substrates. Protease activity has beenimplicated in many disease states.

Cathepsin B is a lysosomal cysteine protease normally involved inproenzyme processing and protein turnover. Elevated levels of activityhave been implicated in tumor metastasis (Sloane, B. et al., “CathepsinB and its endogenous inhibitors: the role in tumor malignancy,” CancerMetastasis Rev. 9:333-352, 1990), rheumatoid arthritis (Werb, Z.“Proteinases and matrix degradation,” in Textbook of Rheumatology,Keller, W. N.; Harris, W. D.; Ruddy, S.; Sledge, C. S., Eds., 1989, W.B. Saunder Co., Philadelphia, Pa., pp. 300-321), and muscular dystrophy(Katunuma N. & Kominami E., “Abnormal expression of lysosomal cysteineproteinases in muscle wasting diseases,” Rev. Physiol. Biochem.Pharmacol. 108:1-20, 1987).

Calpains are cytosolic or membrane bound Ca++-activated proteases whichare responsible for degradation of cytoskeletal proteins in response tochanging calcium levels within the cell. They contribute to tissuedegradation in arthritis and muscular dystrophy (see Wang K. K. & YuenP. W., “Calpain inhibition: an overview of its therapeutic potential,”Trends Pharmacol. Sci. 15:412-419, 1994).

Interleukin Converting Enzyme (ICE) cleaves pro-IL-1 beta to IL-1 beta,a key mediator of inflammation, and therefore inhibitors or ICE mayprove useful in the treatment of arthritis (see, e.g., Miller B. E. etal., “Inhibition of mature IL-1 beta production in murine macrophagesand a murine model of inflammation by WIN 67694, an inhibitor of IL-1beta converting enzyme,” J. Immunol. 154:1331-1338, 1995). ICE orICE-like proteases may also function in apoptosis (programmed celldeath) and therefore play roles in cancer, AIDS, Alzheimer's disease,and other diseases in which disregulated apoptosis is involved (seeBarr, P. J.; Tomei, L. D., “Apoptosis and its Role in Human Disease,”Biotechnol. 12:487-493, 1994).

HIV protease plays a key role in the life cycle of HIV, the AIDS virus.In the final steps of viral maturation it cleaves polyprotein precursorsto the functional enzymes and structural proteins of the virion core.HIV protease inhibitors were quickly identified as an excellenttherapeutic target for AIDS (see Huff, J. R., “HIV protease: a novelchemotherapeutic target for AIDS,” J. Med. Chem. 34:2306-2314, and havealready proven useful in its treatment as evidenced by the recent FDAapproval of ritonavir, Crixivan, and saquinavir.

Angiotensin converting enzyme (ACE) is part of the renin-angiotensinsystem which plays a central role in the regulation of blood pressure.ACE cleaves angiotensin I to the octapeptide angiotensin II, a potentpressor agent due to its vasoconstrictor activity. Inhibition of ACE hasproved therapeutically useful in the treatment or hypertension(Williams, G. H., “Converting-enzyme inhibitors in the treatment ofhypertension,” N. Engl. J. Med. 319:1517-1525, 1989.

Collegenases cleave collagen, the major constituent of the extracellularmatrix (e.g., connective tissue, skin, blood vessels). Elevatedcollagenase activity contributes to arthritis (Krane S. M. et al.,“Mechanisms of matrix degradation in rheumatoid arthritis,” Ann. N.Y.Acad. Sci. 580:340-354, 1990.), tumor metastasis (Flug M. & Kopf-MaierP., “The basement membrane and its involvement in carcinoma cellinvasion,” Acta Anat. Basel 152:69-84, 1995), and other diseasesinvolving the degradation of connective tissue.

Trypsin-like serine proteases form a large and highly selective familyof enzymes involved in hemostasis/coagulation (Davie, E. W. and K.Fujikawa, “Basic mechanisms in blood coagulation,” Ann. Rev. 799-829,1975) and complement activation (Muller-Eberhard, H. J., “Complement,”Ann. Rev. Biochem. 44:697-724, 1975). Sequencing of these proteases hasshown the presence of a homologous trypsin-like core with amino acidinsertions that modify specificity and which are generally responsiblefor interactions with other macromolecular components (Magnusson et al.,“Proteolysis and Physiological Regulation,” Miami winter Symposia11:203-239, 976)

Thrombin, a trypsin-like serine protease, acts to provide limitedpreoloysis, both in the generation of fibrin from fibrinogen and theactivation of the platelet receptor, and thus plays a critical role inthrombosis and hemostasis (Mann, K. G., “The assembly of blood clottingcomplexes on membranes,” Trends Biochem. Sci. 12:229-233, 1987).Thrombin exhibits remarkable specificity in the removal offibrinopeptides A and B of fibrinogen through the selective cleavage ofonly two Arg-Gly bonds of the one-hundred and eighty-one Arg- or Lys-Xaasequences in fibrinogen (Blomback, H., Blood Clotting Enzymology,Seeger, W. H. (ed.), Academic Press, New York, 1967, pp. 143-215).

Many significant disease states are related to abnormal hemostasis,including acute coronary syndromes. Aspirin and heparin are widely usedin the treatment of patients with acute coronary syndromes. However,these agents have several intrinsic limitations. For example, thrombosiscomplicating the rupture of atherosclerotic plaque tends to be athrombin-mediated, platelet-dependent process that is relativelyresistant to inhibition by aspirin and heparin (Fuster et al., “Thepathogenesis of coronary artery disease and the acute coronarysyndromes,” N. Engl. J. Med. 326:242-50, 1992).

Thrombin inhibitors prevent thrombus formation at sites of vascularinjury in vivo. Furthermore, since thrombin is also a potent growthfactor which initiates smooth muscle cell proliferation at sites ofmechanical injury in the coronary artery, inhibitors block thisproliferative smooth muscle cell response and reduce restenosis.Thrombin inhibitors would also induce the inflammatory response invascular wall cells (Harker et al., Am. J. Cardiol 75:12B-16B, 1995).

In view of the important biological role played by the β-sheet, there isa need in the art for compounds which can stabilize the intrinsicβ-sheet structure of a naturally occurring or synthetic peptide, proteinor molecule. There is also a need in the art for making stable β-sheetstructures, as well as the use of such stabilized structures to effector modify biological recognition events which involve β-sheetstructures. The present invention fulfills these needs and providesfurther related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention is directed to β-sheet mimeticsand, more specifically, to β-sheet mimetics which stabilize the β-strandstructure of a natural or synthetic peptide, protein or molecule.

In one aspect of this invention, β-sheet mimetics are disclosedincluding a bicyclic ring system, wherein the β-sheet mimetic has thegeneral structure (I):

and pharmaceutically acceptable salts thereof, wherein R₁, R₂ and R₃ areindependently selected from amino acid side chain moieties andderivatives thereof; A is selected from —C(═)—, —(CH₂)₁₋₄—,—C(═O)(CH₂)₁₋₃—, —(CH₂)₁₋₂O— and —(CH₂)₁₋₂S—; B is selected from N andCH; C is selected from —C(═O)—, —(CH₂)₁₋₃—, —C—, —S—, —O—(CH₂)₁₋₂— and—S(CH₂)₁₋₂—; Y and Z represent the remainder of the molecule; and anytwo adjacent CH groups of the bicyclic ring may form a double bond; wishthe provisos that (i) R₁ is an amino acid side chain moiety orderivative thereof other than hydrogen, (ii) when R₁ is benzyl, R₂ andR₃ are both hydrogen, A is —CH₂CH₂— and B is CH, then C is not —CH₂—,(iii) when R₁ is methyl, R₂ and R₃ are both hydrogen, A is —CH₂O— and Bis CH, then C is not —CH₂—, and (iv) when R₁ is benzyl, R₂ and R₃ areboth hydrogen, A is —CH₂— and B is CH, then C is not —S—.

In one embodiment of structure (I) above, β-sheet mimetics are disclosedhaving the following structure (II):

wherein R₁, R₂ and R₃ are independently selected from amino acid sidechain moieties and derivatives thereof; A is selected from —C(═O)—,—(CH₂)₁₋₄— and —C(═O)(CH₂)₁₋₃—; B is selected from N and CH; C isselected from —C(═C)— and —(CH₂)₁₋₃—; Y and Z represent the remainder ofthe molecule and the bicyclic ring system is saturated (i.e., containsno double bonds between adjacent CH groups of the bicyclic ring system).

In an embodiment of structure (II) where B is CH and R₃ is hydrogen,β-sheet mimetics are disclosed having the following structures (III),(IV) and (V):

wherein R₁ and R₂ are independently selected from amino acid side chainmoieties and derivatives thereof; n is an integer from 1 to 4; p is aninteger from 1 to 3; and Y and Z represent the remainder of themolecule.

In an embodiment o structure (II) where B is N and R₃ is hydrogen,β-sheet mimetics are disclosed having the following structures (VI),(VII) and (VIII):

wherein R₁ and R₂ are independently selected from amino acid side chainmoieties and derivatives thereof; n is an integer from 1 to 4; p is aninteger from 1 to 3; and Y and Z represent the remainder of themolecule.

In preferred embodiments of this aspect of the invention, β-sheetmimetics are disclosed having then following structures (IX), (X) and(XI):

wherein R₁ and R₂ are independently selected from amino acid side chainmoieties and derivatives thereof; n is an integer from 1 to 4; and Y andZ represent the remainder of the molecule.

In a further preferred embodiment of this aspect of the invention, aβ-sheet mimetic is disclosed of structure (X) above wherein n is 2, andhaving the following structure (Xa),

wherein R₁ and R₂ are independently selected from amino acid side chainmoieties and derivatives thereof; and Y and Z represent the remainder ofthe molecule.

In another embodiment of structure (I) above, β-sheet mimetics aredisclosed having the following structure (XII):

wherein R₁, R₂ and R₃ are independently selected from amino acid sidechain moieties and derivatives thereof; A is selected from —(CH₂)₁₋₄—,—(CH₂)₁₋₂O— and —(CH₂)₁₋₂S—, C is selected from —(CH₂)₁₋₃—, —O—, —S—,—O(CH₂)₁₋₂— and —S(CH₂)₁₋₂—; Y and Z represent the remainder of themolecule and the bicyclic ring system is saturated.

In an embodiment of structure (XII) where A is —(CH₂)₁₋₄—, β-sheetmimetics are disclosed having the following structure (XIII):

wherein R₁, R₂ and R₃ are independently selected from amino acid sidechain moieties and derivatives thereof; n is an integer from 1 to 4; Cis selected from —(CH₂)₁₋₃—, —O—, —S—, —O(CH₂)₁₋₂— and —S(CH₂)₁₋₂—; andY and Z represent the remainder of the molecule.

In an embodiment of structure (XII) where A is —(CH₂)₁₋₂O— or—(CH₂)₁₋₂S—, β-sheet mimetics are disclosed having the followingstructures (XIV) and (XV):

wherein R₁, R₂ and R₃ are independently selected from amino acid sidechain moieties and derivatives thereof; m is an integer from 1 to 2; pis an integer from 1 to 3; and Y and Z represent the remainder of themolecule.

In an embodiment of structure (XII) where C is —(CH₂)₁₋₃—, β-sheetmimetics are disclosed having the following structure (XVI):

wherein R₁, R₂ and R₃ are independently selected from an amino acid sidechain moiety and derivatives thereof; p is an integer from 1 to 3; A isselected from —(CH₂)₁₋₄—, —(CH₂)₁₋₂O— and —(CH₂)₁₋₂S—; and Y and Zrepresent the remainder of the molecule.

In an embodiment of structure (XII) where C is —O— or —S—, β-sheetmimetics are disclosed having the following structures (XVII) and(XVIII):

wherein R₁, R₂ and R₃ are independently selected from amino acid sidechain moieties and derivatives thereof; p is an integer from 1 to 3; andY and Z represent the remainder of the molecule.

In an embodiment of structure (XII) where C is —O(CH₂)₁₋₂— or—S(CH₂)₁₋₂—, β-sheet mimetics are disclosed having the followingstructures (XIX) and (XX):

wherein R₁, R₂ and R₃ are independently selected from amino acid sidechain moieties and derivatives thereof; p is an integer from 1 to 3; mis an integer from 1 to 2; and Y and Z represent the remainder of themolecule.

In a further aspect of the present invention, β-sheet modified peptidesor proteins are disclosed wherein a β-sheet mimetic of this invention iscovalently attached to at least one amino acid of a naturally occurringor synthetic peptide or protein. In this embodiment, Y and/or Z in theabove structures (I) through (XX) represent one or more amino acids ofthe peptide or protein. In a related embodiment, a method for impartingand/or stabilizing a β-sheet structure of a natural or synthetic peptideor protein is disclosed. This method includes covalently attaching oneor more β-sheet mimetics of this invention within, or to the end of, apeptide or protein.

In yet a further embodiment, methods are disclosed or inhibiting aprotease in a warm-blooded animal by administering to the animal aneffective amount of a compound of this invention. Proteases includeserine proteases, such as thrombin, elastase and Factor X, as well asaspartic, cysteine and metallo proteases.

Other aspects of this invention will become apparent upon reference tothe following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot showing the effect of various concentrations ofstructure (20b) on platelet deposition in a vascular graft.

FIG. 2 is a plot showing the effect of various concentrations ofstructure (39) on platelet deposition in a vascular graft.

FIG. 3 is a plot showing the effect of various concentrations ofstructure (29b) on platelet deposition in a vascular graft.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the β-sheet is an important structural component formany biological recognition events. The β-sheet mimetics of thisinvention serve to impart and/or stabilize the β-sheet structure of anatural or synthetic peptide, protein or molecule, particularly withregard to conformational stability. In addition, the β-sheet mimetics ofthis invention are more resistant to proteolytic breakdown, thusrendering a peptide, protein or molecule containing the same moreresistant to degradation.

The β-sheet mimetics of this invention are generally represented bystructure (I) above, as well as the more specific embodimentsrepresented by structures (II) through (XX), and have stereochemistriesrepresented by structures (I′) through (I″″) below:

wherein R₁, R₂, R₃, A, B, C, Y and Z are as defined above. In otherwords, all stereoconformations of structure (I), as well as the morespecific embodiments represented by structures (II) through (XX), areincluded within the scope of this invention. For example, the β-sheetmimetics of this invention may be constructed to mimic thethree-dimensional conformation of a β-sheet comprised of naturallyoccurring L-amino acids, as well as the structure of a β-sheet comprisedof one or more D-amino acids. In a preferred embodiment, the β-sheetmimetic has the stereoconformations of structure (I′) or (I″)

As used in the context of this invention, the term “remainder of themolecule” (as represented by Y and Z in structures (II) through (XX)above) may be any chemical moiety. For example, when the β-sheet mimeticis located within he length of a peptide or protein, Y and Z mayrepresent amino acids or the peptide or protein. Alternatively, if twoor more β-sheet mimetics are linked, the Y moiety of a first β-sheetmimetic may represent a second β-sheet mimetic while, conversely, the Zmoiety of the second β-sheet mimetic represents the first β-sheetmimetic. When the β-sheet mimetic is located at the end of a peptide orprotein, or when the β-sheet mimetic is not associated with a peptide orprotein, Y and/or Z may represent a suitable terminating moiety.Representative terminating moieties for the Z moiety include, but arenot limited to, —H, —OH, —R, —C(═O)R and —SO₂R (where R is a C1-C8 alkylor aryl moiety), or may be a suitable protecting group for proteinsynthesis, such as BOC, FMOC or CBZ (i.e., tert-butyloxycarbonyl,9-fluorenylmethoxycarbonyl, and benzyloxycarbonyl, respectively).Similarly, representative terminating moieties for the Y moiety include,but are not limited to, —H, —OH, —R, —NHOH, —NHNHR, —C(═O)OR, —C(═O)NHR,—CH₂Cl,

(where R is a C1-C8 alkyl or aryl moiety), or a heterocyclic moiety,such as pyridine, pyran, thiophan, pyrrole, furan, thiophene, thiazole,benzthiazole, oxazole, benzoxazole, imidazole and benzimidazole.

As used herein, the term “an amino acid side chain moiety” representsany amino acid side chain moiety present in naturally occurringproteins, including (but not limited to) the naturally occurring aminoacid side chain moieties identified in Table 1 below. Other naturallyoccurring side chain moieties of this invention include (but are notlimited to) the side chain moieties of 3,5-dibromotyrosine,3,5-diiodotyrosine, hydroxylysine, naphthylalanine, thienylalanine,γ-carboxyglutamate, phosphotyrosine, phosphoserine and glycosylatedamino acids such as glycosylated serine, asparagine and threonine.

TABLE 1 Amino Acid Side Chain Moiety Amino Acid —H Glycine —CH₃ Alanine—CH(CH₃)₂ Valine —CH₂CH(CH₃)₂ Leucine —CH(CH₃)CH₂CH₃ Isoleucine—(CH₂)₄NH₃ ⁺ Lysine —(CH₂)₃NHC(NH₂)NH₂ ⁺ Arginine

Histidine —CH₂COO⁻ Aspartic acid —CH₂CH₂COO⁻ Glutamic acid —CH₂CONH₂Asparagine —CH₂CH₂CONH₂ Glutamine

Phenylalanine

Tyrosine

Tryptophan —CH₂SH Cysteine —CH₂CH₂SCH₃ Methionine —CH₂OH Serine—CH(OH)CH₃ Threonine

In addition to naturally occurring amino acid side chain moieties, theamino acid side chain moieties of the present invention also includevarious derivatives thereof. As used herein, a “derivative” of an aminoacid side chain moiety includes all modifications and/or variations tonaturally occurring amino acid side chain moieties. For example, theamino acid side chain moieties of alanine, valine, leucine, isoleucineand phenylalanine may generally be classified as lower chain alkyl, arylor aralkyl moieties. Derivatives of amino acid side chain moietiesinclude other straight chain or branched, cyclic or noncyclic,substituted or unsubstituted, saturated or unsaturated lower chainalkyl, aryl or aralkyl moieties.

As used herein, “lower chain alkyl moieties” contain from 1-12 carbonatoms, “lower chain aryl moieties” contain from 6-12 carbon atoms, and“lower chain aralkyl moieties” contain from 7-12 carbon atoms. Thus, inone embodiment, the amino acid side chain derivative is selected from aC₁₋₁₂ alkyl, a C₆₋₁₂ aryl and a C₇₋₁₂ aralkyl, and in a more preferredembodiment, from a C₁₋₇ alkyl, a C₆₋₁₀ aryl and a C₇₋₁₁ aralkyl.

Amino acid side chain derivatives of this invention further includesubstituted derivatives of lower chain alkyl, aryl and aralkyl moieties,wherein the substituent is selected from (but are not limited to) one ormore of the following chemical moieties: —OH, —OR, —COOH, —COOR, —CONH₂,—NH₂, —NHR, —NRR, —SH, —SR, —SO₂R, —SO₂H, —SOR and halogen (including F,Cl, Br and I), wherein each occurrence of R is independently selectedfrom a lower chain alkyl, aryl or aralkyl moiety. Moreover, cyclic lowerchain alkyl, aryl and aralkyl moieties of this invention includenaphthalene, as well as heterocyclic compounds such as thiophene,pyrrole, furan, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline,pyrrolidine, pyridine, pyrimidine, purine, quinoline, isoquinoline andcarbazole. Amino acid side chain derivatives further include heteroalkylderivatives of the alkyl portion of the lower chain alkyl and aralkylmoieties, including (but not limited to) alkyl and aralkyl phosphonatesand silanes.

Bicyclic lactams are known in the art. See., e.g., Columbo, L. et al.,Tet. Lett. 36(4):625-628, 1995; Baldwin, J. E. et al., Heterocycles34(5):903-906, 1992; and Slomczynska, U. et al., J. Org. Chem.61:1198-1204, 1996. However, the bicyclic lactams of the invention arenot disclosed in these references.

As mentioned above, the β-sheet mimetics of this invention serve toimpart and/or stabilize the β-sheet structure of a protein, peptide ormolecule. The β-sheet mimetic may be positioned at either the C-terminusor N-terminus of the protein, peptide or molecule, or it may be locatedwithin the protein, peptide or molecule itself. In addition, more thanone β-sheet mimetic of the present invention may be incorporated in aprotein, peptide or molecule.

The β-sheet mimetics of this invention may be synthesized by a number ofreaction schemes. For example, the various embodiments of structure (I)may be synthesized according to the following reaction schemes (1)through (17).

Reaction Scheme (1)

Structure (III) and representative compounds thereof having structure(IIIa) can be synthesized by the following reaction schemes:

Reaction Scheme (2)

Structure (IV) can be synthesized by the following reaction scheme:

Reaction Scheme (3)

Representative compounds of structure (V) having structure (Va) can besynthesized by the following reaction scheme, where structure (Ia) inscheme (3) is a representative structure of the invention having adouble bond in the bicyclic ring system:

In addition, representative compounds of structure (V) having structure(Vb) may be synthesized by the following reaction scheme, and when A ofstructure (II) is —C(═O) (CH₂)₁₋₃—, a related compound (designated (IIa)below) can be synthesized by the following reaction scheme:

Reaction Scheme (4)

Representative compounds of structure (VI) having structures (VIa) and(VIb) below, wherein R₃ is hydrogen, can be synthesized by the followingreaction scheme (see Holmes and Neel, Tet. Lett. 31:5567-70, 1990):

Representative compounds of structure (II) wherein R₃ is an amino acidside chain moiety or derivative thereof may also be prepared accordingto the above scheme

Reaction Scheme (5)

Representative compounds of structure (VII) having structure (VIIa) canbe synthesized by the following reaction scheme:

Reaction Scheme (6)

Structure (VIII) can be synthesized by the following reaction scheme:

Reaction Scheme (7)

Representative compounds of structure (IX) having structures (IXa) and(IXb) shown below, can be synthesized by the following reaction scheme:

Reaction Scheme (8)

Representative compounds of structure (X) having structures (Xb) and(Xc) can be synthesized by the following reaction scheme (see Perkin, &Sigmund, J. Org. Chem. 52:4007-4013, 1987):

Reaction Scheme (9)

Structure (XI) may be synthesized by the following reaction scheme (seePerkin, J. Chem. Soc. Perk. Trans. 1:155-164, 1984):

Reaction Scheme (10)

Structure (XIII) may be synthesized by the following reaction scheme:

Reaction Scheme (11)

Structures (XIV) and (XV) may be synthesized by the following reactionscheme:

Reaction Scheme (12)

Structure (XVI) may be synthesized by the following reaction scheme:

Reaction Scheme (13)

Structures (XVII) and (XVIII) may be synthesized by the followingreaction scheme:

Reaction Scheme (14)

Structures (XIX) and (XX) may be synthesized by the following reactionscheme:

According to the definition of structure (I) above, the bicyclic ringsystem may contain adjacent CH groups (i.e., the bicyclic ring systemmay be formed, at least in part, by a —CH—CH— group). Compounds whereinsuch a —CH—CH— group is replaced with a —C═C— are also included withinthe scope of structure (I) (i.e., any two adjacent CH groups of thebicyclic ring may together form a double bond). It should be noted thatR₁ according to the definition of structure (I) is a moiety other thanhydrogen. Inspection of structure (I) indicates that the bicyclic ringatom to which R₁ is bonded may not be part of a carbon-carbon doublebond according to structure (I) of the invention. However, R₂ and R₃ maybe hydrogen, and thus the bicyclic ring atoms to which R₂ and/or R₃ arebonded may form part of a carbon-carbon double bond in compounds ofstructure (I).

Reaction Schemes (15), (16) and (17) illustrate synthetic methodologyfor preparing representative compounds of structure (I) wherein thebicyclic ring system is formed in part by a —C═C— group.

In β-sheet mimetics of the invention, preferred Y groups have thestructure:

where a preferred stereochemistry is:

Preferred R₄ groups are organoamine moieties having from about 2 toabout 10 carbon atoms and at least one nitrogen atom. Suitableorganoamine moieties have the chemical formula C₂₋₁₀H₄₋₂₀N₁₋₆O₀₋₂; andpreferably have the chemical formula C₃₋₇H₇₋₁₄N₁₋₄O₀₋₁. Exemplaryorganoamine moieties of the invention are:

In the above structure, R₅ is selected from (a) alkyl of 1 to about 12carbon atoms, optionally substituted with 1-4 of halide, C₁₋₅alkoxy andnitro, (b) —C (═O)NH—C₁₋₅alkyl, wherein the alkyl group is optionallysubstituted with halide or C₁₋₅alkoxy, (c) —C(═O)NH—C₁₋₁₀aralkyl wherethe aryl group may be optionally substituted with up to five groupsindependently selected from nitro, halide, —NH—(C═O)C₁₋₅alkyl,—NH—(C═O)C₆₋₁₀aryl, C₁₋₅alkyl and C₁₋₅alkoxy, and (d) monocyclic andbicyclic heteroaryl of 4 to about 11 ring atoms, where the ring atomsare selected from carbon and the heteroatoms oxygen, nitrogen andsulfur, and where the heteroaryl ring may be optionally substituted withup to about 4 of halide, C₁₋₅alkyl, C₁₋₅alkoxy, —C(═O)NHC₁₋₅alkyl, —C(═O)NHC₆₋₁₀aryl, amino, —C(═O)OC₁₋₅alkyl and —C(═O)OC₆₋₁₀aryl.

Preferred R₅ groups are:

wherein R₆ is hydrogen, nitro, halide, NH—C(═O)—C₁₋₅alkyl,NH—C(═C)—C₆₋₁₀aryl, C₁-C₅alkyl and C₁-C₅ alkoxy;

wherein X is halide;

wherein E is —O—, —NH— or —S— and R₇ and R₈ are independently selectedfrom hydrogen, C₁₋₅alkyl, —C(═O)OC₁₋₅alkyl, —C(═O)OC₅₋₁₀aryl,—C(═O)NHC₁₋₅alkyl and —C(═O) NHC₆₋₁₀aryl; and

wherein E and R₆ are as defined previously.

The β-sheet mimetics of the present invention may be used in standardpeptide synthesis protocols, including automated solid phase peptidesynthesis. Peptide synthesis is a stepwise process where a peptide isformed by elongation of the peptide chain through the stepwise additionof single amino acids. Amino acids are linked to the peptide chainthrough the formation of a peptide (amide) bond. The peptide link isformed by coupling the amino group of the peptide to the carboxylic acidgroup of the amino acid. The peptide is thus synthesized from thecarboxyl terminus to the amino terminus. The individual steps of aminoacid addition are repeated until a peptide (or protein) of desiredlength and amino acid sequence is synthesized.

To accomplish peptide (or protein or molecule) synthesis as describedabove, the amino group of the amino acid to be added to the peptideshould not interfere with peptide bond formation between the amino acidand the peptide (i.e., the coupling of the amino acid's carboxyl groupto the amino group of the peptide). To prevent such interference, theamino groups of the amino acids used in peptide synthesis are protectedwith suitable protecting groups. Typical amino protecting groupsinclude, for example, BOC and FMOC groups. Accordingly, in oneembodiment of the present invention, the β-sheet mimetics of the presentinvention bear a free carboxylic acid group and a protected amino group,and are thus suitable for incorporation into a peptide by standardsynthetic techniques.

The β-sheet mimetics of this invention have broad utility in naturallyoccurring or synthetic peptides, proteins and molecules. For example,the β-sheet mimetics disclosed herein have activity as inhibitors of thelarge family of trypsin-like serine proteases, including thosepreferring arginine or lysine as a P′ substituent. These enzymes areinvolved in blood coagulation, and include (but are not limited to)Factor VIIa, Factor IXa, Factor Xa, thrombin, kallikrein, urokinase(which is also involved in cancer metastasis) and plasmin. Thus, theability to selectively inhibit these enzymes has wide utility intherapeutic applications involving cardiovascular disease and oncology.To this end, the following β-sheet mimetics can be synthesized on solidsupport (e.g., PAM resin):

In the above β-sheet mimetics, L is an optional linker.

The β-sheet mimetics may then be cleaved from the solid support by, forexample, aminolysis, and screened as competitive substrates againstappropriate agents, such as the chromogenic substrate BAPNA(benzyoylarginine paranitroanalide)(see Eichler and Houghten,Biochemistry 32:11035-11041, 1993)(incorporated herein by reference).Alternatively, by employing a suitable linker moiety, such screening maybe performed while the β-sheet mimetics are still attached to the solidsupport.

Once a substrate is selected by the above kinetic analysis, the β-sheetmimetic may be converted into a protease inhibitor by modifications tothe C-terminal—that is, by modification to the Y moiety. For example,the terminal Y moiety may be replaced with —CH₂Cl, —CF₃, —H, or—C(O)NHR. Appropriate R moieties may be selected using a library ofsubstrates, or using a library of inhibitors generated using amodification of the Procedure of Wasserman and Ho (J. Org. Chemn.59:4364-4366, 1994) (incorporated herein by reference).

Libraries of compounds containing β-strand templates may be constructedto determine the optimal sequence for substrate recognition or binding.Representative strategies to use such libraries are discussed below.

A representative β-sheet mimetic substrate library may be constructed asfollows. It should be understood that the following is exemplary ofmethodology that may be used to prepare a β-sheet mimetic substratelibrary, and that other libraries may be prepared in an analogousmanner.

In a first step, a library of the following type:

may be constructed on a solid support (PEGA resin, Meldal, M.Tetrahedron Lett. 33:3077-80, 1992; controlled pore glass, Singh et al.,J. Med. Chem. 38:217-19, 1995). The solid support may then be placed ina dialysis bag (Bednarski et al., J. Am. Chem. Soc. 109:1283-5, 1987)with the enzyme (e.g., a protease) in an appropriate buffer. The bag isthen placed in a beaker with bulk buffer. The enzymatic reaction ismonitored as a function of time by HPLC and materials cleaved from thepolymer are analyzed by MS/MS. This strategy provides informationconcerning the best substrates for a particular enzyme/protease.

The syntheses of the above β-sheet mimetic is illustrated by theretrosynthetic procedure shown next:

The complexity of he library generated by this technique is (R₁) (R₃)(R) (Y) Assuming R₁, R₃ and R are selected from naturally occurringamino acid side chain moieties, n is constant, and Y is H, Ac or —SO₂Ras defined above, a library having on the order of 24,000 members[(20)(20) (20)(3)] is generated.

After screening the library against a protease, the library may thenrecovered and screened with a second protease, and so on.

In addition, a library of inhibitors can be constructed and screened ina standard chromogenic assay. For example, the library may beconstructed as follows, where the following example is merelyrepresentative of the inhibitor libraries that may be prepared in ananalogous manner to the specific example provided below.

(See Wasserman et al., J. Org. Chem. 59:4364-6, 1994.)

A further alternative strategy is to link the library through thesizechain R group as shown below.

A library of aspartic protease inhibitors may be constructed having thefollowing exemplary structure, and then cleaved from the resin andscreened:

Similarly, for metalloproteases, a library having the exemplarystructure shown below may be constructed and then cleaved from the resinto provide a library of hydroxamic acids:

The activity of the β-sheet mimetics of this invention may be furtherillustrated by reference to Table 2 which lists biologically activepeptides. In particular, the peptides of Table 2 are known to havebiological activity as substrates or inhibitors.

Table 2 Biologically Active Protease Inhibitors

(a) (D)FPR (Thrombin) Enzyme 40:144-48, 1988

(b) (D)IEGR (Factor X) Handbook of Synthetic Substrates for theCoagulation and Fibronlytic Systems, H. C. Hemker, pp. 1-175, 1983,Martinus Nijhoff Publishers, The Hague.

In view of the above biologically active peptides, β-sheet mimetics ofthis invention may be substituted for one or more amino acids thereof.For example, the following β-sheet modified peptides may be synthesized:

More generally, the β-sheet mimetics of this invention can besynthesized to mimic any number of biologically active peptides byappropriate choice of the R₁, R₂, R₃, Y and Z moieties (as well as theA, B and C moieties of structure (I) itself). This is furtherillustrated by Table 3 which discloses various modifications which maybe made to the 5-sheet mimetics of structure (I) to yield biologicallyactive compounds. In Table 3, R₂ and R₃ are independently chosen fromamong the atoms or groups shown under the “R₂/R₃” column.

TABLE 3 Modifications to Structure (I) to Yield Biological ActiveCompounds (I)

R₁ R₂/R₃ Y Z I. PROTEASE INHIBITORS A. Serine 1. Thrombin C₆-C₁₀aromatic (e.g., hydrogen hydrogen, alkyl, aryl, phenyl, benzyl,naphthyl), C₁C₁₀ aliphatic

or cycloaliphatic, substituted C₆-C₁₀ aromatic, —SiR₃, —CO₂H, —CO₂R

2. Elastase C₁-C₁₀ aliphatic hydrogen or C₁- C₁₀ heterocyclic

acyl

3. Factor X C₁-C₁₀ aliphatic carboxylic hydrogen

D(Ile) Acyl Dansyl aromatic carboxylate

C₁-C₁₀ acidic heterocyclic

B. Aspartic 1. HIV1 C₁-C₁₀ aliphatic or arginine C₁-C₁₀ aliphatic oracyl

{circle around (1)} = C₁-C₁₀ aliphatic     C₁-C₁₀ aromatic {circlearound (2)} = amino acid     C₁-C₁₀ alkyl     C₁-C₁₀ aryl     acyl    hydrogen C. Cysteine 1. Cathepsin B C₆-C₁₀ aromatic, C₁-C₁₀aliphatic C₁-C₁₀ basic aromatic hydrophobic

benzyl acyl

2. Calpain C₆-C₁₀ aromatic, C₁-C₁₀ aliphatic, hydrophobic C₁-C₁₀aliphatic

benzyl acyl 3. ACE C₁-C₁₀ aliphatic hydrogen

dihydro- cinnamic, aromatic, aliphatic, acetyl

D. Metallo 1. ACE C₁-C₁₀ aliphatic indoyl C₁-C₁₀ aromatic —OH

2. Collagenase C₁-C₁₀ alkyl C₁-C₁₀ aromatic, hydroxyl C₁-C₁₀ aliphatic,C₁-C₁₀ basic

or

C₆-C₁₀ aromatic C₁-C₁₀ alkyl —NHOH hydroxyl C₁-C₁₀ aliphatic

When the β-sheet mimetics of this invention are substituted for one ormore amino acids of a biologically active peptide, the structure of theresulting β-sheet modified peptide (prior to cleavage from the solidsupport, such as PAM) may be represented by the following diagram, whereAA₁ through AA₃ represent the same or different amino acids:

The precise β-sheet mimetic may be chosen by any of a variety oftechniques, including computer modeling, randomization techniques and/orby utilizing natural substrate selection assays. The β-sheet mimetic mayalso be generated by synthesizing a library of β-sheet mimetics, andscreening such library members to identify active members as disclosedabove.

Once the optimized β-sheet mimetic is chosen, modification may then bemade to the various amino acids attached thereto. A series of β-sheetmodified peptides having a variety of amino acid substitutions are thencleaved from the solid support and assayed to identify a preferredsubstrate. It should be understood that the generation of suchsubstrates may involve the synthesis and screening of a number ofβ-sheet modified peptides, wherein each β-sheet modified peptide has avariety of amino acid substitutions in combination with a variety ofdifferent β-sheet mimetics. In addition, it should also be recognizedthat, following cleavage of the β-sheet modified peptide from he solidsupport, the Z moiety is AA₃ and the Y moiety is AA₂ and AA₁ in theabove diagram. (While this diagram is presented for illustration,additional or fewer amino acids may be linked to the β-sheetmimetic—that is, AA₃ may be absent or additional amino acids my bejoined thereto; and AA₂ and/or AA₁ may be omitted or additional aminoacids may be joined thereto).

Once a preferred substrate is identified by the procedures disclosedabove, the substrate may be readily converted to an inhibitor by knowntechniques. For example, the C-terminal amino acid (in this case AA₁)may be modified by addition of a number of moieties known to impartinhibitor activity to a substrate, including (but not limited to) —CF₃(a known reversible serine protease inhibitor, —C₂Cl (a knownirreversible serine protease inhibitor. —CH₂N₂+and —CH₂S(CH₃)₂+(knowncysteinyl protease inhibitors), —NHOH (a known metalloproteaseinhibitor),

(a known cysteinyl protease inhibitor), and

(a known aspartyl protease inhibitor).

While the utility of the β-sheet mimetics of this invention have beendisclosed with regard to certain embodiments, at will be understood thata wide variety and type of compounds can be made which includes theβ-sheet mimetics of the present invention. For example, a β-sheetmimetic of this invention may be substituted for two or more amino acidsof a peptide or protein. In addition to improving and/or modifying theβ-sheet structure of a peptide or protein, especially with regard toconformational stability, the β-sheet mimetics of this invention alsoserve to inhibit proteolytic breakdown. This results in the addedadvantage of peptides or proteins which are less prone to proteolyticbreakdown due to incorporation of the β-sheet mimetics of thisinvention.

In another aspect, the present invention encompasses pharmaceuticalcompositions prepared for storage or administration which comprise atherapeutically effective amount of a β-sheet mimetic or compound of thepresent invention in a pharmaceutically acceptable carrier.Anticoagulant therapy is indicated for the treatment and prevention of avariety of thrombotic conditions, particularly coronary artery andcerebrovascular disease. Those experienced in this field are readilyaware or the circumstances requiring anticoagulant therapy.

The “therapeutically effective amount” of a compound of the presentinvention will depend on the route of administration, the type ofwarm-blooded animal being treated, and the physical characteristics ofthe specific animal under consideration. These factors and theirrelationship to determining this amount are well known to skilledpractitioners in the medical arts. This amount and the method ofadministration can be tailored to achieve optimal efficacy but willdepend on such factors as weight, diet, concurrent medication and otherfactors which as noted hose skilled in the medical arts will recognize.

The “therapeutically effective amount” of the compound of the presentinvention can range broadly depending upon the desired affects and thetherapeutic indication. Typically, dosages will be between about 0.01mg/kg and 100 mg/kg body weight, preferably between about 0.01 and 10mg/kg, body weight.

“Pharmaceutically acceptable carriers” for therapeutic use are wellknown in the pharmaceutical art, and are described, for example, inRemingtons Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaroedit. 1985). For example, sterile saline and phosphate-buffered salineat physiological pH may be used. Preservatives, stabilizers, dyes andeven flavoring agents may be provided in the pharmaceutical composition.For example, sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid may be added as preservatives. In addition, antioxidants andsuspending agents may be used.

Thrombin inhibition is useful not only in the anticoagulant therapy ofindividuals having thrombotic conditions, but is useful wheneverinhibition of blood coagulation is required such as to preventcoagulation of stored whole blood and to prevent coagulation in otherbiological samples for testing or storage. Thus, the thrombin inhibitorscan be added to or contacted with any medium containing or suspected ofcontaining thrombin and in which it is desired that blood coagulation beinhibited (e.g. when contacting the mammal's blood with materialselected from the group consisting of vascular grafts, stems, orthopedicprosthesis, cardiac prosthesis, and extracorporeal circulation systems).

The thrombin inhibitors can be co-administered with suitableanti-coagulation agents or thrombolytic agents such as plasminogenactivators or streptokinase to achieve synergistic effects in thetreatment of various ascular pathologies. For example, thrombininhibitors enhance the efficiency of tissue plasminogenactivator-mediated thrombolytic reperfusion. Thrombin inhibitors may beadministered first following thrombus formation, and tissue plasminogenactivator or other plasminogen activator is administered thereafter.They may also be combined with heparin, aspirin, or warfarin.

The thrombin inhibitors of the invention can be administered in suchoral forms as tablets, capsules (each of which includes sustainedrelease o timed release formulations), pills, powders, granules,elixers, tinctures, suspensions, syrups, and emulsions. Likewise, theymay be administered in intravenous (bolus or infusion), intraperitoneal,subcutaneous, or intramuscular form, all using forms well known to thoseof ordinary skill in the pharmaceutical arts. An effective but non-toxicamount of the compound desired can be employed as an anti-aggregationagent or treating ocular build up of fibrin. The compounds may beadministered intraocularly or topically as well as orally orparenterally.

The thrombin inhibitors can be administered in the form of a depotinjection or implant preparation which may be formulated in such amanner as to permit a sustained release of the active ingredient. Theactive ingredient can be compressed into pellets or small cylinders andimplanted subcutaneously or intramuscularly as depot injections orimplants. Implants may employ inert materials such as biodegradablepolymers or synthetic silicones, for example, Silastic, silicone rubberor other polymers manufactured by the Dow-Corning Corporation.

The thrombin inhibitors can also be administered in the form of liposomedelivery systems, such as small unilamellar vesicles, large unilamellarvesicles and multilamellar vesicles. Liposomes can be formed from avariety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The thrombin Inhibitors may also be delivered by the use of monoclonalantibodies as individual carriers to which the compound molecules arecoupled. The thrombin inhibitors may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinlypyrrolidone, pyran copolymer,polyhydroxy-propylmethacrylamide-phenol,polyhydroxyethyl-aspartarnide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the thrombininhibitors may be coupled to a class of biodegradable polymers useful inachieving controlled release of a drug, for example, polylactic acid,polyglycolic acid, copolymers of polylactic and polyglycolic acid,polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters,polyacetals, polydibydropyrans, polycyanoacrylates and cross linked oramphipathic block copolymers of hydrogels.

The dose and method of administration can be tailored to achieve optimalefficacy but will depend on such factors as weight, diet, concurrentmedication and other factors which those skilled in the medical artswill recognize. When administration is to be parenteral, such asintravenous on a daily basis, injectable pharmaceutical compositions canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to infection, or as emulsions.

Tablets suitable or oral administration of active compounds o: theinvention (e.g., structures (47), (20b), (37), (39), (29a), (35), (45),(51), (29b), (41) and (13b)), can be prepared as follows:

Amount-mg Active Compound 25.0 50.0 100.0 Microcrystalline 37.25 100.0200.0 cellulose Modified food corn 37.25 4.25 8.5 starch Magnesiumstearate 0.50 0.75 1.5

All of the active compound, cellulose, and a portion of the corn starchare mixed and granulated to 10% corn starch paste. The resultinggranulation is sieved, dried and blended with the remainder of the cornstarch and the magnesium stearate. The resulting granulation is thencompressed into tablets containing 25.0, 50.0, and 100.0 mg,respectively, of active ingredient per tablet.

An intravenous dosage form of the above-indicated active compounds maybe prepared as follows:

Active Compound 0.5-10.0 mg Sodium Citrate 5-50 mg Citric Acid 1-15 mgSodium Chloride 1-8 mg Water for q.s. to 1 ml Injection (USP)

Utilizing the above quantities, the active compound is dissolved at roomtemperature in a previously prepared solution of sodium chloride, citricacid, and sodium citrate in Water for Injection (USP, see page 1636 ofUnited States Pharmacopoeia/National Formulary for 1995, published byUnited States Pharmacopoeia Convention, Inc., Rockville, Md., copyright1994).

Compounds of the present invention when made and selected as disclosedare useful as potent inhibitors of thrombin in vitro and in vivo. Assuch, these compounds are useful as in vitro diagnostic reagents toprevent the clotting of blood and as in vivo pharmaceutical agents toprevent thrombosis in mammals suspected of having a conditioncharacterized by abnormal thrombosis.

The compounds of the present invention are useful as in vitro diagnosticreagents for inhibiting clotting in blood drawing tubes. The use ofstoppered test tubes having a vacuum therein as a means to draw bloodobtained by venipuncture into the tube is well known in the medical arts(Kasten, B. L., “Specimen Collection,” Laboratory Test Handbook, 2ndEdition, Lexi-Comp Inc., Cleveland pp. 16-17, Edits. Jacobs, D. S. etal. 1990). Such vacuum tubes may be free of clot-inhibiting additives,in which case, they are useful for the isolation of mammalian serum fromthe blood they may alternatively contain clot-inhibiting additives (suchas heparin salts, EDTA salts, citrate salts or oxalate salts), in whichcase, they are useful for the isolation of mammalian plasma from theblood. The compounds of the present invention are potent inhibitors offactor Xa or thrombin, and as such, can be incorporated into bloodcollection tubes to prevent clotting of the mammalian blood drawn intothem.

The compounds of the present invention are used alone, in combination ofother compounds of the present invention, or in. combination with otherknown inhibitors of clotting, in the blood collection tubes. The amountto be added to such tubes is that amount sufficient to inhibit theformation of a clot when mammalian blood is drawn into the tube. Theaddition of the compounds to such tubes may be accomplished by methodswell known in the art, such as by introduction of a liquid compositionthereof, as a solid composition thereof, or liquid composition which islyophilized to a solid. The compounds of the present invention are addedto blood collection tubes in such amounts that, when combined with 2 to10 mL of mammalian blood, the concentration of such compounds will besufficient to inhibit clot formation. Typically, the requiredconcentration will be about 1 to 10,000 nM, with 10 to 1000 nM beingpreferred.

The following examples are offered by way of illustration, notlimitation.

EXAMPLES Example 1 Synthesis of Representative β-Sheet Mimetic

This example illustrates the synthesis of a representative β-sheetmimetic of this invention.

Synthesis of Structure (1)

Phenylalanine benzaldimine, structure (1), was synthesized as follows.To a mixture of L-phenylalanine methyl ester hydrochloride (7.19 g, 33.3mmol) and benzaldehyde (3.4 ml, 33.5 mmol) stirred in CH₂Cl₂ (150 ml) atroom temperature was added triethylamine (7.0 ml, 50 mmol). Anhydrousmagnesium sulfate (2 g) was added to the resulting solution and themixture was stirred for 14 h then filtered through a 1 inch pad ofCelite with CH₂Cl₂. The filtrate was concentrated under reduced pressureto ca. one half of its initial volume then diluted with an equal volumeof hexanes. The mixture was extracted twice with saturated aqueousNaHCO₃, H₂O and brine then dried over anhydrous Na₂SO₄ and filtered.Concentration of the filtrate under vacuum yielded 8.32 g (93% yield) ofcolorless oil. ¹H NMR analysis indicated nearly pure (>95%)phenylalanine benzaldimine. The crude product was used without furtherpurification.

Synthesis of Structure (2)

α-Allylphenylalanine benzaldimine, structure (2), was synthesized asfollows. To a solution of diisopropylamine (4.3 ml, 33 mmol) stirred inTHF (150 ml) at −78° C. was added dopwise a solution of n-butyllithium(13 ml of a 2.5 M hexane solution, 33 mmol). The resulting solution wasstirred for 20 min. then a solution of phenylalanine benzaldimine (7.97g, 29.8 mmol) in THF (30 ml) was slowly added. The resulting darkred-orange solution was stirred for 15 min. then allyl bromide (3.1 ml,36 mmol) was added. The pale yellow solution was stirred for 30 mm. at−78° C. then allowed to warm to room temperature and stirred anadditional 1 h. Saturated aqueous ammonium chloride was added and themixture was poured into ethyl acetate. The organic phase was separatedand washed with water and brine then dried over anhydrous sodium sulfateand filtered. Concentration of the filtrate under vacuum yielded 8.54 gof a viscous yellow oil. Purification by column chromatography yielded7.93 g (87%.) of α-allylphenylalanine benzaldimine as a viscouscolorless oil.

Synthesis of Structure (3)

α-Allylphenylalanine hydrochloride, structure (3), was synthesized asfollows. To a solution of α-allylphenylalanine benzaldimine (5.94 g,19.3 mmol) stirred In methanol (50 ml) was added 5% aqueous hydrochloricacid (10 ml). The solution was stirred at room temperature for 2 h thenconcentrated under vacuum to an orange-brown caramel. The crude productwas dissolved in CHCl₃ (10 ml) and the solution was heated to boiling.Hexanes (˜150 ml) were added and the slightly cloudy mixture was allowedto cool. The liquid was decanted away from the crystallized solid thenthe solid was rinsed with hexanes and collected. Removal of residualsolvents under vacuum yielded 3.56 g (72%) of pure α-allylphenylalaninehydrochloride as a white crystalline solid.

¹H NMR (500 MHz, CDCl₃) δ 8.86 (3 H, br s), 7.32-7.26 (5H, m), 6.06 (1H,dddd, J=17.5, 10.5, 7.6, 7.3 Hz), 5.33 (1H, d, J=17.5 Hz), 5.30 (1H, d,J=10.5 Hz), 3.70 (3H, s), 3.41 (1H, d, J=14.1 Hz), 3.35 (1H, d, J=14.1Hz), 2.98 (1H, dd, J=14.5, 7.3 Hz), 2.88 (1H, dd, J=14.5, 7.6 Hz).

Synthesis of Structure (4)

N-tert-butyloxycarbonyl-α-allylphenylalanine, structure (4) wassynthesized as follows. To a solution of D,L α-allylphenylalaninehydrochloride (565 mg, 2.21 mmol) stirred in a mixture of THF (15 ml)and water (5 ml) was added di-tert-butyl dicabonate followed by carefuladdition of solid sodium bicarbonate in small portions. The resultingtwo phase mixture was vigorously stirred at room temperature for 2 daysthen diluted with ethyl acetate. The organic phase was separated andwashed with water and brine then dried over anhydrous sodium sulfate andfiltered. Concentration of the filtrate under vacuum yielded a colorlessoil that was purified by column chromatography (5 to 10% EtCAc inhexanes gradient elution) to yield 596 mg (86%) ofN-tert-butyloxycarbonyl-α-allylphenylalanine.

TLC R_(f)=0.70 (silica, 20% EtOAc in hexanes); ¹H NMR (500 MHz, CDCl₃) δ7.26-7.21 (3H, m), 7.05 (2H, d, J=6.1 Hz), 5.64 (1H, dddd, J=14.8, 7.6,7.2, 7.2 Hz), 5.33 (1H, b s), 5.12-5.08 (2H, m), 3.75 (3H, s), 3.61 (1H,d, J=13.5 Hz), 3.21 (1H, dd, J=13.7, 7.2 Hz), 3.11 (1H, d, J=13.5 Hz),2.59 (1H, dd, J=13.7, 7.6 Hz), 1.47 (9H, s).

Synthesis of Structure (5)

An aldehyde of structure (5) was synthesized as follows. Ozone wasbubbled through a solution of 2.10 g (6.57 mmol) of the structure (4)olefin stirred at −78° C. in a mixture of CH₂Cl₂ (50 ml) and methanol(15 ml) until the solution was distinctly blue in color. The solutionwas stirred an additional 15 min. then dimethyl sulfide was slowlyadded. The resulting colorless solution was stirred at −78° C. for 10min. then allowed to warm to room temperature and stirred for 6 h. Thesolution was concentrated under vacuum to 2.72 g of viscous pale yellowoil which was purified by column chromatography (10 to 20% EtOAc inhexanes gradient elution) to yield 1.63 g of pure aldehyde as a viscouscolorless oil.

TLC R_(f)=0.3 (silica, 20% EtOAc in hexanes); ¹H NMR (500 MHz, CDCl₃) δ9.69 (1H, br s), 7.30-7.25 (3H, m,), 7.02 (2H, m,), 5.56 (1H, br s),3.87 (1H, d, J=17.7 Hz,), 3.75 (3 H, s,), 3.63 (1H, d, J=13.2 Hz), 3.08(1H, d, J=17.7 Hz), 2.98 (1H, d, J=13.2 Hz,), 1.46 (9H, s,).

Synthesis of Structure (6)

A hydrazone of structure (6) was synthesized as follows. To a solutionof the aldehyde of structure (5) (1.62 g, 5.03 mmol) stirred in THF (50ml) at room temperature was added hydrazine hydrate (0.32 ml, 6.5 mmol).The resulting solution was stirred at room temperature for 10 min. thenheated to reflux for 3 days. The solution was allowed to cool to roomtemperature then concentrated under vacuum to 1.59 g (105% crude yield)of colorless foam. The crude hydrazone product, structure (6), was usedwithout purification.

TLC R_(f)=0.7 (50% EtOAc in hexanes); ¹H NMR (500 MHz, CDCl₃) δ 8.55(1H, br s), 7.32-7.26 (3H, m), 7.17 (1H, br s), 7.09 (2H, m), 5.55 (1H,br s), 3.45 (1H, d, J=17.7 Hz), 3.29 (1H, d, J=13.5 Hz), 2.90 (1H, d,J=13.5 Hz), 2.88 (1H, dd, J=17.7, 1.3 Hz), 1.46 (9H, s); MS (CI+, NH₃)m/z 304.1 (M+H⁺).

Synthesis of Structure (7)

A cyclic hydrazide of structure (7) was synthesized as follows. Thecrude hydrazone of structure (6) (55 mg, 0.18 mmol) and platinum oxide(5 mg, 0.02 mmol) were taken up in methanol and the flask was fittedwith a three-way stopcock attached to a rubber balloon. The flask wasflushed with hydrogen gas three times, the balloon was inflated withhydrogen, and the mixture was stirred vigorously under a hydrogenatmosphere for 17 hours. The mixture was filtered through Celite withethyl acetate and the filtrate was concentrated under vacuum to a whiteform. Purification of the white foam by flash chromatography yielded 44mg of the pure cyclic hydrazide of structure (7) (80%).

¹H NMR (500 MHz, CDCl₃) δ 7.34-7.28 (3H, m), 7.21 (2H, m), 6.95 (1H, brs), 5.29 (1H, br s), 3.91 (1H, br s), 3.35 (1H, d, J=12.9 Hz), 3.00 (1H,ddd, J=13.9, 5.3, 5.0 Hz), 2.96 (1H, d, J=12.9 Hz), 2.67 (1H, br m),2.38 (1H, br m), 2.30 (1H, ddd, J=13.9, 5.4, 5.0 Hz), 1.45 (9H, s); MS(CI+, NH₃) m/z 306.2 (M+H⁺).

Synthesis of Structure (8)

Structure (8) was synthesized as follows. To a solution of the cyclichydrazide of structure (7) (4.07 g, 13.32 mmol) stirred in ethylacrylate (200 ml) at 90° C. was added formaldehyde (1.2 mL of a 37%aqueous solution). The mixture was heated to reflux for 15 h thenallowed to cool to room temperature and concentrated under vacuum to awhite foam. The products were separated by column chromatography (5%then 10% acetone/chloroform) to yield 0.851 g of the least polardiastereomer of the bicyclic ester, structure (8b), and a more polardiastereomer (8a). The impure fractions were subjected to a secondchromatography to afford more pure structure (8b), 25% combined yield.

¹H NMR (500 MHz, CDCl₃) δ 7.27-7.21 (3H, m), 7.09 (2H, d, J=6.5 Hz), 5.9(1 H, br s), 4.52 (1 H, dd, J=9.1, 3.4 Hz), 4.2 (2H, m), 3.40 (1 H, d,J=12.5 Hz), 3.32 (1H, d, J=12.5 Hz), 3.10 (2 H, m), 2.79 (1H, br m),2.66 (1H, br m), 2.79 (1H, br m), 2.66 (1H, br m), 2.54 (1H, br m), 2.46(1H, m), 2.18 (1H, m), 1.44 (9H, s), 1.28 (3H, t, J=7.0 Hz); MS (CI+,NH₃) 418.4 (M+H⁺).

Synthesis of Structure (9b)

Structure (9b) was synthesized as follows. To a solution of the leastpolar ethyl ester (i.e., structure (8b)) (31 mg, 0.074 mmol) stirred inTHF (1 ml) was added aqueous lithium hydroxide (1 M, 0.15 ml). Theresulting mixture was stirred at room temperature for 2 h then thereaction was quenched with 5% aqueous citric acid. The mixture wasextracted with ethyl acetate (2×) then the combined extracts were washedwith water and brine. The organic layer was dried over anhydrous sodiumsulfate, filtered and concentrated under vacuum to a colorless glass.The crude acid, structure (9b), was used in subsequent experimentswithout further purification.

Synthesis of Structure (10b)

Structure (10b) was synthesized as follows. The crude acid of structure(9b) (30 mg, 0.074 mmol), HArg(PMC)pNA (41 mg, 0.074 mmol), and HOBt (15mg, 0.098 mmol) were dissolved in THF (1 ml) then diisopropylethylamine(0.026 ml, 0.15 mmol) was added followed by EDC (16 mg, 0.084 mmol). Theresulting mixture was stirred at room temperature for 4 h then dilutedwith ethyl acetate and extracted with 5% aqueous citric acid, saturatedaqueous sodium bicarbonate, water and brine. The organic layer was driedover anhydrous sodium sulfate, filtered and concentrated under vacuum to54 mg of pale yellow glass. The products were separated by columnchromatography to yield 33 mg (50%) of a mixture of diastereomers of thecoupled (i.e., protected) product, structure (10b). MS (CI+, NH₃) m/z566.6 (M+H⁺).

Synthesis of Structure (11b)

A β-sheet mimetic of structure (11b) was synthesized as follows. Asolution of 0.25 ml of H₂O, 0.125 ml of 1,2-ethanedithiol and 360 mg ofphenol in 5 ml of TFA was prepared and the protected product ofstructure (10b) (33 mg, 0.035 mmol) was dissolved in 2 ml of thissolution. The resulting solution was stirred a room temperature for 3 hthen concentrated under reduced pressure. Ether was added to theconcentrate and the resulting precipitate was collected bycentrifugation. The precipitate was triturated with ether andcentrifuged two more times then dried in a vacuum desiccator for 14 h.The crude product (14 mg) was purified by HPLC chromatography to yieldthe β-sheet mimetic of structure (11b). MS (CI+, NH₃) m/z 954.8 (M+Na⁺).

Synthesis of Structure (12b)

Structure (12b) was synthesized as follows. To a solution of the crudeacid of structure (9b) (24 mg, 0.062 mmol) and N-methylmorpholine (0.008ml), stirred in THF (1 ml) at −50° C. was added isobutyl chloroformate.The resulting cloudy mixture was stirred for 10 min. then 0.016 ml (0.14mmol) of N-methylmorpholine was added followed by a solution ofHArg(Mtr)CH₂Cl (50 mg, 0.068 mmol) in THF (0.5 ml). The mixture was keptat −50° C. for 20 min. then was allowed to warm to room temperatureduring 1 h. The mixture was diluted with ethyl acetate and extractedwith 5% aqueous citric acid, saturated aqueous sodium bicarbonate andbrine. The organic layer was dried over anhydrous sodium sulfate,filtered and concentrated under vacuum to yield 49 mg of colorlessglass, structure (12). Separation by column chromatography yielded 12 mgof a less polar diastereomer and 16 mg of a more polar diastereomer.

¹H NMR (500 MHz, CDCl₃) δ 7.93 (1H, br s), 7.39-7.31 (3H, m), 7.16 (2H,d, J=6.9 Hz), 6.52 (1H, s), 6.30 (1H, br s), 5.27 (1H, s), 4.74 (1H, dd,J=9.1, 6.9 Hz), 4.42 (1H, br d, J=6.8 Hz), 4.33 (1H, d, J=6.8 Hz), 3.82(3H, s), 3.28 (1H, d, J=13.3 Hz), 3.26-3.12 (4H, m), 2.98 (1H, d, J=13.3Hz), 2.69 (3H, s), 2.60 (3H, s), 2.59-2.33 (4H, m), 2.25-2.10 (3H, m),2.11 (3H, s), 1.77 (1H, br m), 1.70-1.55 (3H, br m), 1.32 (9H, s).

Synthesis of Structure (13b)

A β-sheet mimetic of structure (13b) was synthesized as follows. Themore polar diastereomer of structure (12b) (16 mg, 0.021 mmol) wasdissolved in 95% TFA/H₂O (1 ml) and the resulting solution was stirredat room temperature for 6 h then concentrated under vacuum to 11 mg ofcrude material. The crude product was triturated with ether and theprecipitate was washed twice with ether then dried under high vacuum for14 h. ¹H NMR analysis indicated a 1:1 mixture of fully deprotectedproduct and product containing the Mtr protecting group. The mixture wasdissolved in 95% TFA/H₂O and stirred for 2 days and the product wasrecovered as above. Purification of the product by HPLC yielded 5 mg ofthe pure compound of structure (13b). MS (EI+) m/z 477.9 (M⁺).

Example 2 Synthesis of Representative β-Sheet Mimetic

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (14)

N,O-Dimethyl hydroxamate, structure (14), was synthesized as follows. Toa mixture ofBoc-N^(g)-4-methoxy-2,3,6-trimethylbenzenesulfonyl-L-arginine (8.26 g,14.38 mmol), N,O-dimethylhydroxylamine hydrochloride (2.78 g, 28.5 mmol)and 1-hydroxybenzotriazole hydrate (2.45 g, 16.0 mmol) stirred in THF(150 ml) at ambient temperature was added N,N-diisopropylethylamine (7.5ml, 43 mmol) followed by solid EDC (3.01 g, 15.7 mmol). The resultingsolution was stirred for 16h then diluted with ethyl acetate (200 ml)and extracted sequent ally with 5% aqueous citric acid, saturatedaqueous sodium bicarbonate, water and brine. The organic solution wasdried over anhydrous sodium sulfate and Filtered. Concentration of thefiltrate under vacuum yielded 7.412 g of white foam.

¹H NMR (500 Mhz, CDCl₃): δ 6.52 (1H, s), 6.17 (1H, br s), 5.49 (1H, d,J=8.8 Hz), 4.64 (1H, br t), 3.82 (3H, s), 3.72 (3H, s), 3.36 (1H, br m),3.18 (3H, s), 3.17 (1H, br m), 2.69 (3H, s), 2.61 (3H, s), 2.12 (3H, 2),1.85-1.55 (5H, m), 1.41 (9H, s); MS (FB+): m/z 530.5 (M+H⁺).

Synthesis of Structure (15)

Structure (15) was synthesized as follows. To a solution of the arginineamide (7.412 g, 13.99 mmol) stirred in dichloromethane (150 ml) at roomtemperature was added N,N-diisopropylethylamine (2.9 ml, 17 mmol)followed by di-tert-butyldicarbonate (3.5 ml, 15.4 mmol) andN,N-dimethylaminopyridine (0.175 g, 1.43 mmol). The water. The aqueouslayer was separated and extracted with two 100 ml portions ofdichloromethane. The combine extracts were shaken with brine then driedover anhydrous sodium sulfate and filtered. Concentration of thefiltrate under vacuum yielded a white foam that was purified by flashchromatography to yield 8.372 g of white foam.

¹H NMR (500 MHz, CDCl₃): δ 9.79 (1H, s), 8.30 (1H, t, J=4.96), 6.54 (1H,s), 5.18 (1H, d, J=9.16 Hz), 4.64 (1H, m), 3.83 (3H, s), 3.74 (3 H, s),3.28 (2H, dd, J=12.6, 6.9 Hz), 3.18 (3H, s), 2.70 (3H, s), 2.62 (3H, s),2.14 (3H, s), 1.73-1.50 (5H, m), 1.48 (9H, s), 1.42 (9H, s); MS (FB+):m/z 630.6 (M+H⁺).

Synthesis of Structure (16)

The arginal, structure (16), was synthesized as follows. To a solutionof the arginine amide structure (15) stirred in toluene at −78° C. undera dry argon atmosphere was added a solution of diisobutylaluminumhydride in toluene (1.0 M, 7.3 ml) dropwise over a period of 15 minutes.The resulting solution was stirred for 30 minutes then a second portionof diisobutylaluminum hydride (3.5 ml) was added and stirring wascontinued for 15 minutes. Methanol (3 ml) was added dropwise and thesolution was stirred at −78° C. for 10 minutes then allowed to warm toroom temperature. The mixture was diluted with ethyl acetate (100 ml)and stirred vigorously with 50 ml of saturated aqueous potassium sodiumtartrate for 2.5 h. The aqueous phase was separated and extracted withethyl acetate (2×100 ml). The extracts were combined with the originalorganic solution and shaken with brine then dried over anhydrous sodiumsulfate and filtered. Concentration of the filtrate under vacuum yieldeda white foam that was separated by flash chromatography to yield 1.617 gof the aldehyde as a white foam.

¹H NMR (500 MHz, CDCl₃): δ 9.82 (1H, s), 9.47 (1H, s), 8.35 (1H, br t),6.55 (1H, s), 5.07 (1H, d, J=6.9 Hz), 4.18 (1H, br m), 3.84 (3H, s),3.25 (2H, m), 2.70 (3H, s), 2.62 (3H, 5), 2.14 (3H, s), 1.89 (1H, m),1.63-1.55 (4H, m), 1.49 (9H, s), 1.44 (9H, s); MS (FB+): m/z 571.6(M+H⁺).

Synthesis of Structure (17)

Hydroxybenzothiazole, structure (17) was synthesized as follows. To asolution of benzothiazole (1.55 ml 14 mmol) stirred in anhydrous diethylether (60 ml) at −78° C. under a dry argon atmosphere was added asolution of n-butyllithium (2.3 M in hexane, 5.6 ml, 14 mmol) dropwiseover a period of 10 minutes. The resulting orange solution was stirredfor 45 minutes then a solution of the arginal structure (16) (1.609 g,2.819 mmol) in diethyl ether (5 ml) was slowly added. The solution wasstirred for 1.5 h then saturated aqueous ammonium chloride solution wasadded and the mixture was allowed to warm to room temperature. Themixture was extracted with ethyl acetate (3×100 ml) and the combinedextracts were extracted with water and brine then dried over anhydroussodium sulfate and filtered. Concentration of the filtrate under vacuumyielded a yellow oil that was purified by flash chromatography (30% then40% ethyl acetate/hexanes eluent) to yield 1.22 g of thehydroxybenzothiazoles (ca. 2:1 mixture of diastereomers) as a whitefoam.

The mixture of hydroxybenzothiazoles (1.003 g, 1.414 mmol) was stirredin CH₂Cl₂ (12 ml) at room temperature and trifluoroacetic acid (3 ml)was added. The resulting solution was stirred for 1.5 h thenconcentrated under reduced pressure to yield 1.22 g of thebenzothiazolylarginol trifluoroacetic acid salt as a yellow foam.

MS (EI+): m/z 506.2 (M+H⁺).

Synthesis of Structure (18b)

The bicyclic compound, structure (18b) was synthesized as follows. Thebicyclic acid of structure (9b) from Example 1 (151 mg, 0.387 mmol) andHOBt hydrate (71 mg, 0.46 mmol) were dissolved in THF (5 ml) anddiisopropylethylamine (0.34 ml, 1.9 mmol) was added followed by EDC (89mg, 0.46 mmol). After stirring for ten minutes a solution of thebenzothiazolylarginol trifluoroacetic acid salt (structure (17) 273 mg,0.372 mmol) in THF (1 ml) was added along with a THE (0.5 ml) rinse. Themixture was stirred at room temperature for 15 h then diluted with ethylacetate and extracted sequentially with 5% aqueous citric acid,saturated aqueous sodium bicarbonate, water and brine. The organicsolution was dried over anhydrous sodium sulfate, filtered andconcentrated under vacuum to 297 mg of a yellow glass. ¹H NMR analysisindicated a mixture of four diastereomeric amides which includedstructure (18b).

MS (ES+): m/z 877 (M⁺).

Synthesis of Structure (19b)

Structure (19b) was synthesized as follows. The crudehydroxybenzothiazole (247 mg, 0.282 mmol) was dissolved in CH₂Cl₂ (5 ml)and Dess-Martin periodinane (241 mg, 0.588 mmol) was added. The mixturewas stirred at room temperature for 6 h then diluted with ethyl acetateand stirred vigorously with 10% aqueous sodium thiosulfate for 10minutes. The organic solution was separated and extracted with saturatedaqueous sodium bicarbonate, water and brine then dried over anhydroussodium sulfate and filtered. Concentration of the filtrate under vacuumyielded 252 mg of yellow glass. ¹H NMR analysis indicated a mixture oftwo diastereomeric ketobenzothiazoles which included structure (19b).

Synthesis of Structure (20b)

The ketobenzothiazole, structure (20), was synthesized as follows.Ketobenzothiazole (19) (41 mg, 0.047 mmol) was dissolved in 95% aqueoustrifluoroacetic (0.95 ml) acid and thioanisole (0.05 ml) was added. Theresulting dark solution was stirred for 30 hours at room temperaturethen concentrated under vacuum to a dark brown gum. The gum wastriturated with diethyl ether and centrifuged. The solution was removedand the solid remaining was triturated and collected as above two moretimes. The yellow solid was dried in a vacuum desiccator for 2 hoursthen purified by HPLC (Vydac reverse phase C-4 column (22×250 mm ID).Mobile phase: A=0.05% TFA in water; B=0.05% TFA in acetonitrile. Theflow rate was 10.0 mL/min. The gradient used was 8% B to 22% B over 25min, and isochratic at 22% thereafter. The peak of interest (structure(20b)) eluted at 42 minutes) to give 2.5 mg of the deprotected product,structure (20b).

MS (ES+): 563.5 (M+H⁺).

Example 3 Activity of a Representative α-Sheet Mimetic as a ProteolyticSubstrate

This example illustrates the ability of a representative β-sheet mimeticof this invention to selectively serve as a substrate for thrombin andFactor VII. The β-sheet mimetic of structure (11b) above was synthesizedaccording the procedures disclosed in Example 1, and used in thisexperiment without further modification.

Both the thrombin and Factor VII assays of this experiment were carriedout at 37° C. using a Hitachi UV/Vis spectrophotometer (model U-3000).Structure (11b) was dissolved in deionized water. The concentration wasdetermined from the absorbance at 342 nm. Extinction coefficient of 8270liters/mol/cm was employed. The rate of structure (11b) hydrolysis wasdetermined from the change in absorbance at 405 nm using an extinctioncoefficient for p-nitroaniline of 9920 liters/mol/cm for reactionbuffers. Initial velocities were calculated from the initial linearportion of the reaction progress curve. Kinetic parameters weredetermined by unweighted nonlinear least-squares fitting of the simpleMichaelis-Menten equation to the experimental data using GraFit (Version3.0, Erithacus Software Limited).

For the thrombin assay, experiments were performed in pH 8.4 Tris buffer(Tris, 0.05M; NaCl, 0.15M). 6.4 NIH units of bovine thrombin (fromSigma) were dissolved into 10 ml of the assay buffer to yield 10 nMthrombin solution. In a UV cuvette, 130 to 148 μl of the buffer and 100μl of the thrombin solutions were added, preincubated at 37° C. for 2minutes, and finally 2 to 20 microliters (to make the final volume at250 μl ) of 0.24 mM structure (11b) solution was added to initiate thereaction. The first two minutes of the reactions were recorded forinitial velocity determination. Eight structure (11b) concentrationpoints were collected to obtain the kinetic parameters. k_(cat) andK_(M) were calculated to be 50 s⁻¹ and 3 μM, respectively. k_(cat)/K_(M)was found to be 1.67×10⁷ M⁻¹ s⁻¹.

For the Factor VII assay, pH 8.0 Tris buffer (0.05 M Tris, 5 mM CaCl₂,0.15 M NaCl, 0.1% TWEEN 20, 0.1% BSA) was used. 10 μl of 20 μM humanFactor VIIa (FVIIa) and 22 μM of human tissue factor (TF) was brought toassay buffer to make 160 nM FVIIa and TF solutions, respectively. 40 to48 μl of buffer, 25 μl of FVIIa and 25 μl TF solution were added to acuvette, and incubated at 37° C. for 5 minutes, then 2 to 10 μl of 2.4mM structure (11b) solution was added to the cuvette o initiate reaction(final volume was 100 ml). The initial 3 minutes reaction progresscurves were recorded. Five structure (11b) concentration points werecollected. The initial rates were linear least-square fitted against theconcentrations of structure (11b) with GraFit. The k_(cat)/K_(M) wascalculated from the slope and found to be 17,500 M⁻¹s⁻¹.

In both the thrombin and Factor VII assay of this experiment, (D)FPR-PNAwas run as a control. Activity of structure (11b) compared to thecontrol was 0.76 and 1.38 for thrombin and Factor VII, respectively(Factor VII: K_(cat)/K_(M)=1.27×10⁴ M⁻¹ s⁻¹; thrombin:K_(cat)/K_(M)=2.20×10⁷ M⁻¹ S⁻¹).

Example 4 Activity of a Representative β-Sheet Mimetic as a ProteaseInhibitor

This example illustrates the ability of a representative β-sheet mimeticof this invention to function as a protease inhibitor for thrombin,Factor VII, Factor X, urokinase, tissue plasminogen activator (t-PA),protein C, plasmin and trypsin. The β-sheet mimetic of structure (13b)above was synthesized according to the procedures disclosed in example1, and used in this experiment.

All inhibition assays of this experiment were performed a roomtemperature in 96 well microplates using a Bio-Rad microplate reader(Model 3550). 0.29 mg of structure (13b) was dissolved into 200 ml of0.02 N hydrochloric acid deionized water solution. This solution (2.05mM) served as the stock solution for all the inhibition assays. Thehydrolysis of chromogenic substrates was monitored at 405 nm. Thereaction progress curves were recorded by reading the plates typically90 times with 30 seconds to 2 minute intervals. The initial rate weredetermined by unweighted nonlinear least-squares fitting to a firstorder reaction in GraFit. The determined initial velocities were thennonlinear least-square fitted against the concentrations of structure(13b) using GraFit to obtain IC₅₀. Typically, eight structure (13b)concentration points were employed for IC₅₀ determination.

For the thrombin assay, N-c-tosyl-Gly-Pro-Arg-pNA (from Sigma) was usedat 0.5 mM concentration in 1% DMSO (v/v) pH 8.4 Tris buffer assubstrate. From structure (13b) stock solution two steps or dilutionwere made. First, 1:2000 dilution into 0.02 N hydrochloride solution,then 1:100 dilution into pH 8.4 Tris buffer. The final dilution ofstructure (13b) served as the first point (10 nM). Seven sequentialdilutions were made from the first point with a dilution factor of 2.Into each reaction well, 100 μl of 10 nM thrombin solution and 50 μl ofstructure (13b) solution was added. The mixture of the enzyme andinhibitor was incubated for 20 minutes, then 100 μl of 0.5 mM substratesolution was added to initiate the reaction. The IC₅₀ of structure (13b)against thrombin was found to be 1.2±0.2 nM.

In the Factor VII assay, S-2288 (from Pharmacia), D-Ile-Pro-Arg-pNA wasused at 20 μM in deionized water as substrate. From the stock ofstructure (13b), a 1:100 dilution was made into pH 8.0 Tris buffer. Thisdilution served as she first point of the inhibitor (20 μM). From thisconcentration point 6 more sequential dilutions were made with adilution factor of 2. 50 μl of 16 nM FVIIa and TF complex solution and40 μl of the inhibitor solutions were added into each well, the mixtureswere incubated for 20 minutes before 10 μl of 20 mM S-2288 was added.IC₅₀ of structure (13b) against factor VII was found to be 140±3 nM.

In the Factor X assay, buffer and substrate are the same as used forthrombin assay. A 1:100 dilution was made into pH 8.4 Tris buffer toserve as the first point. Seven dilutions with a dilution factor of 2were made. The assay protocol is the same as for thrombin except 25 nMof bovine factor Xa (from Sigma) in pH 8.4 Tris buffer was used insteadof thrombin. IC₅₀ of structure (13b) against factor X was found to be385±17 nM.

In the urokinase assay, buffer was pH 8.8 0.05 M Tris and 0.05 M NaCl indeionized water. S-2444 (from Sigma), pyroGlu-Gly-Arg-pNA at 0.5 mM inwater was utilized as substrate. The same dilution procedure was used asfor Factor VII and Factor X. Assay protocol is the same as for thrombinexcept 18.5 nM of human urokinase (from Sigma) was utilized. IC₅₀ wasfound to be 927±138

Tissue Plasminogen Activator (t-PA): Buffer, substrate and the dilutionscheme of structure (13b) were the same as utilized for Factor VIIassay.

Activated Protein C (aPC): Buffer was the same as used in thrombinassay. 1.25 mM S-2366 in the assay buffer was utilized as substrate.Dilutions of structure (13b) were the same as in urokinase assay.

Plasmin: Buffer (see thrombin assay); S-2551 (from Pharmacia),D-Val-Leu-Lys-pNA at 1.25 mM in assay buffer was utilized as substrate.For dilutions of structure (13b) (see urokinase assay).

In the trypsin assay, pH 7.8 Tris (0.10 M Tris and 0.02 M CaCl₂) wasutilized as the buffer. BAPNA (from Sigma) was used at 1 mg/ml in 1%DMSO (v/v) deionized water solution as substrate. The same dilutions ofstructure (13b) were made as for Factor VII assay. 40 μl of 50 μg/mlbovine trypsin (from Sigma) and 20 μl of structure (13b) solution wereadded to a reaction well, the mixture was incubated for 5 minutes before40 μl of 1 mg/ml BAPNA was added to initiate the reaction. The IC₅₀ ofstructure (13b) against trypsin was found to be 160±8 nM.

In the above assays, (D)FPR-CH₂Cl (“PPACK”) was run as a control.Activity of structure (13b) compared to the control was enhanced (seeTable 4).

TABLE 4 IC₅₀ (nM) Enzymes PPACK Structure (13b) Thrombin 1.5 1.2 FactorVII 200 140 Factor X 165 385 Protein C 281 528 Plasmin 699 978 Trypsin212 16 Urokinase 508 927 t-PA 106 632

With respect to prothrombin time (PT), this was determined by incubating(30 minutes at 37° C.) 100 μl of control plasma (from Sigma) with 1-5 μlof buffer (0.05 M Tris, 0.15 M NaCl, pH=8.4) or test compound (i.e.,PPACK or structure (13b)) in buffer. Then 200 μl of prewarmed (at 37° C.for ˜10 minutes) thromboplastin with calcium (from Sigma, was rapidlyadded into the plasma sample. The time required to form clot wasmanually recorded with a stop watch (see Table 5), and was found to becomparable with PPACK.

TABLE 5 PT (second) Concentration PPACK Structure (13b)  0 (Control) 1313  1 pM — 13  10 pM — 17  50 pM — 18 100 pM — 23 200 pM — 24 500 pM 1527  1 nM 18 30  10 nM 22 31  20 nM 25 —  30 nM — 31  40 nM 28 —  50 nM —30  60 nM 30 —  80 nM 31 33

Example 5 Activity of a Representative β-Sheet Mimetic as a ProteaseInhibitor

This example illustrates the ability of a further representative β-sheetmimetic of this invention to function as an inhibitor for thrombin,Factor VII, Factor X, urokinase, Tissue Plasminogen Activator, ActivatedProtein C, plasmin, tryptase and trypsin. The β-sheet mimetic ofstructure (20b) above was synthesized according to the Proceduresdisclosed in Example 2, and used in this experiment.

All inhibition assays were performed at room temperature in 96 wellmicroplates using Bio-Rad microplate reader (Model 3550). A 1 mMsolution of structure (20b) in water served as the stock solution forall the inhibition assays. The hydrolysis of chromogenic substrates wasmonitored at 405 nm. The reaction progress. curves were recorded byreading the plates, typically 60 times with 30 second to 2 minuteintervals. Initial rates were determined by unweighted nonlinearleast-squares fitting to a first order reaction in GraFit (ErithacusSoftware Limited, London, England). The determined initial velocitieswere then nonlinear least-square fitted against the concentrations ofstructure (20b) using GraFit to obtain Ki. The general format of theseassays are: 100 ml of a substrate solution and 100 ml of structure (20b)solution were added in a microplate well, then 50 ml of enzyme solutionwas added to initiate the reaction. Typically, eight structure (20b)concentration points were employed for Ki determination. The values ofKi of structure (20b) against nine serine proteases are tabulated inTable 6.

Thrombin: N-p-tosyl-Gly-Pro-Arg-pNA (from Sigma) was used at 0.5 mMconcentration in 1% DMSO (v/v) pH8.0 tris buffer (tris, 50 mM, TWEEN 20,0.1%, BSA, 0.1%, NaCl, 0.15 M, CaCl₂, 5 mM) as substrate. From structure(20b) stock solution two steps of dilution were made, first, 1:100dilution in water, then 1:50 dilution in the pH8.0 tris buffer to serveas the first point (200 nM). Seven sequential dilutions were made fromthe first point for the assay.

Factor VII: S-2288 (from Pharmacia), D-Ile-Pro-Arg-pNA was used at 2.05mM in the pH 8.0 tris buffer (see thrombin assay). From the stock ofstructure (20b), a 1:100 dilution was made in the tris buffer. From thisconcentration point seven more sequential dilutions were made for theassay.

Factor X: Buffer and substrate were the same as used for thrombin assay.A 1:100 dilution was made in the pH8.0 tris buffer to serve as the firspoint. Seven more dilutions from the first were made for the assay.

Urokinase: Buffer, 50 mM tris, 50 mM NaCl, pH=8.8. S-2444 (from Sigma),pyroGlu-Gly-Arg-pNA at 0.25 mM in buffer was utilized as substrate. 1:10dilution in buffer was made from the stock of structure (20b) as thefirst point, then seven more dilutions from the first point were madefor the assay.

Tissue Plasminogen Activator (t-PA): Buffer, substrate and the dilutionscheme of structure (20b) were the same as utilized for Factor VIIassay.

Activated Protein C (aPC): Buffer was the same as used in thrombinassay. 1.25 my S-2366 in the assay buffer was utilized as substrate.Dilutions of structure (20b) were the same as in urokinase assay.

Plasmin: Buffer (see thrombin assay); S-2251 (from Pharmacia),D-Val-Leu-Lys-pNA at 1.25 mM in assay buffer was utilized as substrate.For dilutions of structure (20b) (see urokinase assay).

Tryptase: 0.1 M tris, 0.2 M NaCl, 0.1 mg/ml heparin, pH=8.0 was utilizedas buffer. 0.5 mM S-2366 (from Pharmacia), L-pyroGlu-Pro-Arg-pNA inbuffer was used as substrate. From the 1 mM stock of structure (20b), 10mM solution was made in water, then 1 mM solution was made in bufferfrom the 10 mM solution to serve as the first concentration point. Fromthis point seven more dilutions were made for the assay.

Trypsin: Buffer, substrate and the dilution scheme of structure (20b)were the same as used for thrombin.

TABLE 6 K₁ (nM) Assay Structure Enzyme Source Conc. (nM) (20b) thrombinbovine plasma 2 0.66 factor VII human 4 270 factor X bovine plasma 8 966urokinase human kidney 3.7 600 t-PA human 10 495 APC human plasma 1 3320plasmin bovine plasma 4 415 tryptase human lung 2 12.4 trypsin bovine 50.64 pancreas

As illustrated by the data presented in Table 6 above, structure (20b)functioned as a good thrombin inhibitor, with good specificity againstfibrinolytic enzymes.

Example 6 Synthesis of Representative β-Sheet Mimetic

This example illustrates the synthesis of a representative β-sheetmimetic of this invention having the following structure (21):

Structure (21) was synthesized as follows. A solution of 48 mg (0.859mmol) N^(a)-FMOC-N^(e)-Cbz-a-ethanal-Lys-Ome [synthesized fromN^(e)-Cbz-Lys-OMe by the same method used for the preparation ofstructure (5) from Phe-OMe], 15.9 mg (0.0859 mmol) Cys-OEt.HCl , and13.2 μL (0.0945 mmol) TEA were in 0.43 mL CH₂Cl₂ were stirred under Arfor 2 hr at room temperature. Bis(bis(trimethylsilyl)amino)tin(II) (39.8μL) was added and the reaction stirred overnight. The reaction solutionwas diluted with 10 mL EtOAc and washed with 6 mL each 10% citrate,water, and brine. The organic layer was dried over Na₂SO₄, filtered, andconcentrated. The resulting residue was purified by flash chromatographyon silica gel using 40% EtOAc/hexanes to give, after drying in vacuo,12.9 mg of colorless oil (23%) as a mixture of diastereomers by ¹H NMR(CDCl₃). MS ES (+) m/z 658.2 (MH⁺, 30), 675.3 (M+Na⁺, 100), 696.1 (M+K⁺,45).

Example 7 Synthesis of Representative β-Sheet Mimetic

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (22)

Structure (22) was synthesized as follows. To a stirred solution ofCbz-Glu(OBn)-OH (5 g, 13.5 mmol) with DMAP (270 mg) and methanol (3 ml)in dichloromethane (100 ml) was added EDCI (3 g) at 0° C. After stirringat 0° C. for 3 h, the solution was starred at room temperature (rt)overnight. After concentration, the residue was taken up into EtOAc (100ml) and 1N HCl (100 ml). The aqueous phase was separated and extractedwith EtOAc (100 ml). The combined organic extracts were washed with sat.NaHCO₃ (100 ml), brine (100 ml), dried (MgSO₄), passed through a shortpad of silica gel, and concentrated to provide 4.95 g an oil (95%). Theproduct was pure enough to use for the next reaction without any furtherpurification. ¹H NMR (CDCl₃) δ 2.00 (m, 1H), 2.25 (m, 1H), 2.50 (m, 2H),3.74 (s, 3H, OCH₃), 4.42 (m, 1H, CHNH), 5.10 and 5.11 (two s, 4H,CH₂Ph), 5.40 (d, 1H, NH), 7.35 (s, 10H, phenyls); MS CI (isobutane) m/z386 (M+H⁺).

Synthesis of Structure (23)

Structure (23) was synthesized as follows: To a stirred solution ofL-Glu-OH (4.41 g, 30 mmol) with triethylamine (8.4 mL, 60 mmol) in1,4-dioxane (40 ml) and H₂O (20 ml) was added Boc₂O (7 g, 32 mmol) atrt. After stirring for 1.5 h, he solution was acidified with 6N HCl (pH2), and extracted with EtOAc (3×100 ml). The combined organic extractswere washed with H₂O (100 ml), brine (50 ml), dried (Na₂SO₄), andconcentrated to provide an oil (9.5 g). Without further purification,the oil was used in the next reaction.

A mixture of above oil (9.5 g) with paraformaldehyde (5 g) andp-TsOH.H₂O (400 mg) in 1,2-dichloromethane (200 ml) was heated at refluxwith a Dean-Stark condenser, which was filled with molecular sieve 4A,for 6 h. After addition of EtOAc (100 ml) and sat. NaHCO₃ (50 ml), thesolution was extracted with sat. NaHCO₃ (3×50 ml). The combined aqueousextracts were acidified with 6N HCl (pH 2), and extracted with EtOAc(3×100 ml). The combined organic extracts were washed with brine (100ml), dried (Na₂SO₄), and concentrated to provide an oil. The crude oilwas purified by flash chromatography (hexane:EtOAc=80:20 to 70:30 to60:40) to provide an oil (4.04 g, 52%) which solidified slowly uponstanding. ¹H NMR (CDCl₃) δ 1.49 (s, 9H, C(CH₃)₃), 2.13 (m, 1H, —CH₂CH₂),2.29 (m, 1H, CH₂CH₂), 2.52 (m, 2H, —CH₂CH₂—), 4.33 (m, 1H, NHCHCH₂),5.16 (d, 1H, J=4.5 Hz, NCH₂O), 5.50 (br, 1H, NCH₂O); ¹³C NMR (CDCl₃) δ25.85, 28.29, 29.33, 54.16, 79.10, 82.69, 152.47, 172.37, 178.13; MS(ES+) m/z 260 (M+H⁺), 282 (M+Na⁺), 298 (M+K⁺)

Synthesis of Structure (24)

Structure (24) was synthesized as follows. To a stirred solution of1,1,1,3,3,3-hexamethyldisilazane (2.1 ml, 10 mmol) in THF (10 ml) wasadded n-BuLi (4 ml of 2.5M in hexane, 10 mmol) at 0° C. The resultingsolution was stirred at the same temperature for 30 min. After coolingto −78° C., to this stirred solution was added a solution of carboxylicacid (23) (1.02 g, 3.94 mmol) in THF (10 ml) followed by rinsings of theaddition syringe with 5 ml THF. The resulting solution was stirred at−78° C. for 1 h, and PhCH₂Br (0.46 ml, 3.9 mmol) was added. Afterstirring at ˜30° C. for 3 h, to this solution was added 1N HCl (50 ml)and the resulting solution was extracted with EtOAc (100 ml). Theorganic extract was washed with brine (50 ml), dried (Na₂SO₄), andconcentrated to provide an oil. The crude product was purified by flashchromatography (hexane:EtOAc=80:20 to 60:40 to 50:50) to provide a foamysolid (1.35 g, 98%); ¹H NMR (CDCl₃) δ 1.55 and 1.63 (two s, 9H, ratio1.5:1 by rotamer, OC(CH₃)₃), 2.2-2.4 (m, 3H, —CH₂CH₂—), 2.6-2.9 (set ofm, 1H, —CH₂CH₂—), 3.04 (d, 1H, J=13.5 Hz, —CH₂Ph), 3.33 and 3.58 (two d,1H, J=13 Hz, ratio 2:1, —CH₂Ph), 4.03 (two d, 1H, J=4 Hz, A of ABq,—NCH₂O—), 4.96 (two d, 1H, J=4 Hz, B of ABq, —NCH₂O—); MS (ES−) m/z 348(M−H⁺).

Synthesis of Structure (25)

Synthesis of structure (25) was carried out as follows. To a stirredsolution of carboxylic acid (24) (1.05 g, 3.0 mmol) in dry THF (5 ml)was added 1,1′-carbonyldiimidazole (500 mg, 3.1 mmol) at rt. Theresulting solution was stirred at rt for 30 min. The solution of acylimidazole was used for the next reaction without purification.

Meanwhile, to a stirred solution of 1,1,1,3,3,3-hexamethyldisilazane(1.6 ml, 7.5 mmol) in THF (5 ml) was added n-BuLi (3 ml of 2.5 Msolution in hexane, 7.5 mmol) at 0° C. After stirring at the sametemperature or 30 min, the solution was cooled to −78° C. To the stirredsolution was added a solution of Cbz-Glu(OBn)-OMe (1.16 g, 3 mmol) inTHF (5 ml) followed by rinsings of the addition syringe with 2 ml THF.The resulting solution was stirred at the same temperature for 15 min.To this stirred solution was added the above acyl imidazole in 3 ml THF.After stirring 30 min. at −78° C., to this solution was added sat. NH₄Cl(50 ml) and extracted with EtOAc (2×75 ml). The combined organicextracts were washed with sat. NaHCO₃ (50 ml), brine (50 ml), dried(Na₂SO₄), passed through a short pad of silica gel, and concentrated toprovide an oil. The crude product was purified by flash chromatography(hexane:EtOAc 90:10 to 80:20 to 70:30 to 60:40) to provide an oil (1.48g, 69%): MS (ES+) m/z 734.4 (M+NH₄ ⁺).

Synthesis of Structure (26a)

Structure (26a) was synthesized as follows. A stirred solution of abovestarting keto ester (25) (530 mg, 0.7 mmol) in EtCH/AcOH (10/1 ml) wastreated with 10% Pd/C (ca. 100 mg) under 20 atm pressure of H₂ for 2days. After filtration through a short pad of Celite, the filtrate wasconcentrated and dissolved in EtOAc (50 ml) The solution was washed with1N HCl (30 ml), sat. NaHCO₃ (30 ml), brine (30 ml), dried (Na₂SO₄), andconcentrated to provide an oil. The crude product was purified by flashchromatography (hexane:EtOAc=80:20 to 60:40 to 50:50 to 20:80 to 0:100)to provide a foamy solid (95 mg, 34%). TLC (EtOAc) R_(f)=0.68; NMR(CDCl₃) δ 1.38 (two s, 9H, OC(CH₃)₃), 1.63 (s, 1H), 1.75 (m, 2H), 2.05(m, 5H), 2.1-2.3 (set of m, 1H), 3.00 (d, 1H, J=14 Hz, CH₂Ph), 3.21 (d,1H, J=13.5 Hz, CH₂Ph), 3.74 (collapsed two s, 4H, OCH₃ and NCH), 4.53(d, 1H, J=9.5 Hz), 5.01 (br, 1H, NH); MS (ES+) m/z 403 (M+H⁺), 425(M+Na⁺). Stereochemistry was assigned by 2D NMR.

Synthesis of Structure (27a)

Structure (27a) was synthesized as follows. To a solution of 28 mg(0.070 mmol) of the bicyclic ester (26a) stirred in 1 ml THF at roomtemperature was added 0.14 ml 1.0 M aqueous lithium hydroxide solution.The mixture was stirred vigorously for 20 h then quenched with 5%aqueous citric acid (1 ml). The mixture was extracted with ethyl acetate(3×25 ml) then the combined extracts were washed with water and brineand dried over anhydrous sodium sulfate. Filtration and concentration ofthe filtrate under vacuum gave 26 mg of white foam, used without furtherpurification.

Synthesis of Structure (28a)

Structure (28a) was synthesized as follows. The bicyclic acid (27a) (26mg, 0.067 mmol), benzothiazolylarginol trifluoroacetic acid salt(structure (17) 61 mg, 0.083 mmol) EDC (21 mg, 0.11 mmol) and HOBthydrate (16 mg, 0.10 mmol) were dissolved in THF (5 ml) anddiisopropylethylamine (0.34 ml, 1.9 mmol) was added. The mixture wasstirred at room temperature for 15 h then diluted with ethyl acetate andextracted sequentially with 5% aqueous citric acid, saturated aqueoussodium bicarbonate, water and brine. The organic solution was dried overanhydrous sodium sulfate, filtered and concentrated under vacuum to 60mg of a yellow glass. ¹H NMR analysis indicated a mixture of fourdiastereomeric amides. MS (ES+): m/z 898 (M+Na⁺).

Synthesis of Structure (29a)

A β-sheet mimetic of structure (29a) was synthesized as follows. Thecrude hydroxybenzothiazole (28a) (60 mg, 0.068 mmol) was dissolved inCH₂Cl₂ (2 ml) and Dess-Martin periodinane (58 mg, 0.14 mmol) was added.The mixture was stirred at room temperature for 6 h then diluted withethyl acetate and stirred vigorously with 10% aqueous sodium thiosulfatefor 10 minutes. The organic solution was separated and extracted withsaturated aqueous sodium bicarbonate, water and brine then dried overanhydrous sodium sulfate and filtered. Concentration of the filtrateunder vacuum yielded 42 mg of yellow glass. ¹H NMR analysis indicated amixture of two diastereomeric ketobenzothiazoles.

The ketobenzothiazole (42 mg, 0.048 mmol) was dissolved in 95% aqueoustrifluoroacetic (0.95 ml) acid and thioanisole (0.05 ml) was added. Theresulting dark solution was stirred for 18 hours at room temperaturethen concentrated under vacuum to a dark brown gum. The gum wastriturated with diethyl ether and centrifuged. The solution was removedand the solid remaining was triturated and collected as above two moretimes. The yellow solid was dried in a vacuum desiccator for 2 hoursthen purified by HPLC to give 1.4 mg of the deprotected product. MS(ES+): 562.4 (M+H⁺). HPLC: (t_(R)=21.17 min.)

Synthesis of Structure (26b)

Structure (26b) was synthesized as follows. A stirred solution of abovestarting keto ester (25) (615 mg, 0.86 mmol) in MeOH/AcOH (10/1 ml) wastreated with 10% Pd/C (ca. 60 mg) under 20 atm pressure of H₂ for 3days. After filtration through a short pad of Celite, the filtrate wasconcentrated to provide an oil. The crude product was purified by flashchromatography (hexane:EtOAc=80:20 to 60:40 to 50:50 to 0:100) tocollect the more polar fraction (50 mg). Rf 0.12 (hexane:EtOAc=60:40);MS (ES+) m/z 4.33 (M+H+).

Above oil was treated with p-TsOH H₂O (5 mg) in 1,2-dichloroethane (10ml) at reflux temperature for 2 days. After concentration, the oilyproduct was purified by preparative TLC (hexane:EtOAc=80:20 to 60:40) togive an oil (10 mg). TLC Rf 0.36 (hexane:EtOAc=60:40); ¹H NMR (CDCl₃) δ1.43 (s, 9H), 1.66 (m, 3H), 1.89 (m, 3H), 2.14 (m, 1H), 2.75 (m, 1H),2.98 (m, 1H, CHN), 3.72 (s, 3H, Me), 4.30 (m, 1H), 5.59 (d, 1H, NH),7.1-7.3 (m, 5H, phenyl); MS CI (NH₃) 403.2 (M+H+). Stereochemistry wasassigned by 2D NMR.

Synthesis of Structure (28b)

Structure (28b) was synthesized as follows. To a solution of 12 mg(0.030 mmol) of the bicyclic ester (26b) stirred in THF 1 ml at roomtemperature was added 0.060 ml 1.0 M aqueous lithium hydroxide solution.The mixture was stirred vigorously for 25 h then quenched with 5%aqueous citric acid (1 ml). The mixture was extracted with ethyl acetate(3×25 ml) then the combined extracts were washed with water and brineand dried over anhydrous sodium sulfate. Filtration and concentration ofthe filtrate under vacuum gave 19 mg of white foam.

The foam, benzothiazolylarginol trifluoroacetic acid salt (30 mg, 0.041mmol) EDC (10 mg, 0.052 mmol) and HOBt hydrate (9 mg, 0.059 mmol) weredissolved in THF (2 ml) and diisopropylethylamine (0.026 ml, 0.15 mmol)was added. The mixture was stirred at room temperature for 30 h thendiluted with ethyl acetate and extracted sequentially with 5% aqueouscitric acid, saturated aqueous sodium bicarbonate, water and brine. Theorganic solution was dried over anhydrous sodium sulfate, filtered andconcentrated under vacuum to 28 mg of a yellow glass. ¹NMR analysisindicated a mixture of four diastereomeric amides. MS (ES+): m/z 898(M+Na⁺).

Synthesis of Structure (29b)

Structure (29b) was synthesized as follows. The crudehydroxybenzothiazole (28b) (28 mg) was dissolved in CH₂Cl₂ (2 ml) andDess-Martin periodinane (29 mg, 0.071 mmol) was added. The mixture wasstirred at room temperature for 18 h then diluted with ethyl acetate andstirred vigorously with 10% aqueous sodium thiosulfate for 10 minutes.The organic solution was separated and extracted with saturated aqueoussodium bicarbonate, water and brine then dried over anhydrous sodiumsulfate and filtered. Concentration of the filtrate under vacuum yielded32 mg of yellow glass. ¹H NMR analysis indicated a mixture of twodiastereomeric ketobenzothiazoles.

The ketobenzothiazole (32 mg) was dissolved in 95% aqueoustrifluoroacetic (0.95 ml) acid and thioanisole (0.05 ml) was added. Theresulting dark solution was stirred for 20 hours at room temperaturethen concentrated under vacuum to a dark brown gum. The gum wastriturated with diethyl ether and centrifuged. The solution was removedand the remaining solid was triturated and collected as above two moretimes. The yellow solid was died in a vacuum desiccator for 2 hours thenpurified by HPLC to give 1.3 mg of the deprotected product. MS (FB+):562.36 (M+H⁺); HPLC: t_(R)=21.51 min. (Gradient 0 to 90% 0.1% TFA inCH₃CN/0.1% TFA in H₂O over 40 min.)

Example 8 Activity of Representative β-Sheet Mimetic as a ProteaseInhibitor

This example illustrates the ability of a further representative β-sheetmimetic of this invention to function as an inhibitor for thrombin,Factor VII, Factor X, Factor X, and trypsin. The β-sheet mimetics ofstructures (29a) and (29b) above were synthesized according to theprocedures disclosed in Example 7, and used in this experiment.

The proteinase inhibitor assays were performed as described in Example 5except as described below for Factor X:. The results are presented inTable 7.

Factor XI. The same buffer was utilized in this assay as in the thrombinassay. 1 mM S-2366 (from Pharmacia), L-pyroGlu-Pro-Arg-pNA, solution inwater was used as substrate. From a 1 mM stock solution of structure(29a) or (29b) in water, a 1:10 dilution was made in buffer. From this100 μM solution, seven serial 1:5 dilutions were made in buffer forassay.

TABLE 7 K₁ (nM) Enzymes Structure (29a) Structure (29b) Thrombin 10.40.085 Trypsin 0.54 0.20 Factor VII 1800 — Factor X 4600 17 Factor XI 391—

Example 9 Activities of Representative β-Sheet Mimetics as a ProteaseInhibitor

This example illustrates the ability of further representative β-sheetmimetics of this invention to function as an inhibitor for thrombin,Factor VII, Factor X, Factor X, tryptase, aPC, plasmin, tPA, urokinaseand trypsin. The β-sheet mimetics of structures (20) and (29b) abovewere synthesized according to the procedures disclosed in Examples 2 and7, respectively, and used in this experiment.

The proteinase inhibitor assays were performed as described in Example 5except as described in Example 8 for Factor XI. The results arepresented in Table 8.

TABLE 8 Structure (20b) Structure (29b)

Selectivity Selectivity Ki (nM) * Ki (nM) * Thrombin 0.65 1 0.085 1Trypsin 0.62 0.95 0.23 2.7 Factor VII 270 415 200 2353 Factor X 222 34219.3 227 Factor XI 27.0 42 75.3 886 Tryptase 12.3 18.9 9.0 106 aPC 33205108 1250 14706 Plasmin 415 638 251 2953 tPA 495 762 92.9 1093 Urokinase600 923 335 3941 *selectivity is the ratio of Ki of an enzyme to the Kiof thrombin

Example 10 Synthesis of Representative β-Sheet Mimetics

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (30)

Structure (30) was synthesized as follows. n-Butyllithium (700 μL, 1.75mmol, 2.5M in hexanes) was added over 5 min to a solution oftris(methylthio)methane (256 μL, 1.95 mmol) in THF (1 ml) at −78° C. Themixture was stirred for 40 min then treated with a solution ofbis-Boc-argininal (structure (16) from Example 2) (100 mg, 1.75 mmol) in2 ml THF, dropwise, over a period of 5 min. After stirring or 1.5 h, thereaction was quenched with saturated NH₄Cl solution and allowed to warmto room temperature. The layers were separated and the aqueous layerextracted with EtOAc (3×), washed with brine (1×), dried (Na₂SO₄) andconcentrated. Purification by flash chromatography (EtOAc:Hexane 1:4)yielded 93 mg (73%) of the orthothiomethyl ester (structure (30)) and 8mg of recovered aldehyde (structure (16)). ¹H NMR (500 MHz, CDCl₃.) δ9.80 (s, 1H), 8.32 (t, J=5.0 Hz, 1H), 6.54 (s, 1H), 5.23 (d, J=9.0 Hz,1H), 4.0 (m, 1H), 3.84 (s, 3H), 3.64 (br s, 1H), 3.38 (br s, 1H), 3.31(m, 2H), 2.70 (s, 3H), 2.62 (s, 3H), 2.19 (s, 9H), 2.14 (s, 3H),1.68-1.50 (m, 4H), 1.49 (s, 9H), 1.43 (s, 9H).

Synthesis of Structure (31)

Structure (31) was synthesized as follows. A mixture of 77 mg (0.11mmol) of the orthothiomethyl ester (structure (30)), 117 mg (0.43 mmol)of mercuric chloride, and 39 mg (0.18 mmol) of mercuric oxide in 2.5 mlof 12:1 methanol/water was stirred at rt for 4 h. The mixture wasfiltered through Celite and the residue washed with EtOAc (3×). Thefiltrate was diluted with water and extracted with EtOAc (3×). Theorganic layer was washed twice with 75% NH₄OAc/NH₄Cl, then with NH₄Cland dried (Na₂SO₄). The solvent was removed in vacuo and the residuepurified by flash chromatography (EtOAc/Hex, 1:3) to give 48 mg (72%) ofthe two diastereomers of structure (31) in a 1:2.7 ratio. ¹H NMR (500MHz, CDCl₃) (major diastereomer) δ 9.80 (s, 1H), 8.33 (t, J=5.0 Hz, 1H),6.54 (s, 1H), 4.66 (d, J=10.5 Hz, 1H), 4.08 (dd, J=5.0, 2.0 Hz, 1H),3.97 (m, 1H), 3.84 (s, 3H), 3.77 (s, 3H), 3.30 (m, 2H), 3.06 (d, J=5.0Hz, 1H), 2.70 (s, 3H), 2.63 (s, 3H), 2.14 (s, 3H), 1.68-1.50 (m, 4H),1.49 (s, 9H), 1.40 (s, 9H); MS (ES+) m/z 631.5 (M+H⁺).

Synthesis of Structure (32)

Structure (32) was synthesized as follows. A solution of 32 mg of themethyl ester (structure (31)) (0.051 mmol) in THF/water (4 m, 1:3) wastreated with 5 mg (0.119 mmol) of LiOH.H₂O. After stirring for 45 min,the reaction was diluted with 5% citric acid and extracted with ethylacetate (3×). The combined extracts were washed with brine, dried overNa₂SO₄ and concentrated to give 30 mg (96%) of structure (32) as a whitesolid. The product was used without further purification. ¹H NMR 500MHz, CDCl₃) δ 9.80 (br s, 1H), 8.29 (br s, 1H), 6.54 (s, 1H), 5.62 (brs, 1H), 4.08 (m, 1H), 3.82 (s, 3H), 3.27 (br s, 3H), 2.69 (s, 3H), 2.62(s, 3H), 2.13 (s, 3H), 1.65-1.50 (m, 4H), 1.48 (s, 9H), 1.37 (s, 9H); MS(ES−) m/z 615.5 (M−H⁺).

Synthesis of Structure (33)

Structure (33) was synthesized as follows. To a solution of the compoundof structure (32) (29 g, 0 047 mmol), HOBt (8 mg, 0.056 mmol) and EDC(11 mg, 0.056 mmol) in THF (5 ml), phenethylamine (7 ml, 0.056 mmol) wasadded followed by diisopropylethylamine (12 μL, 0.071 mmol). Thereaction mixture was stirred at rt overnight and diluted with 5% citricacid. The organic layer was separated and the aqueous phase extractedwith EtOAc (3×) The combined extracts were washed with a saturatedsolution of NaHCO₃, brine, dried over Na₂SO₄, and filtered. Afterconcentration the crude product was purified by chromatography(EtOAc/Hex, 1:1) to give 26 mg (77%) of structure (33) over two steps.¹H NMR (500 MHz, CDCl₃) δ 9.84 (s, 1H), 8.34 (t, J=5 Hz, 1H), 7.28 (m,3H), 7.21 (m, 2H), 7.04 (m, 1H), 6.55 (s, 1H), 5.16 (d, J=8.5 Hz, 1H),4.56 (d, J=5 Hz, 1H), 4.11 (dd, J=5.0, 3.0 Hz, 1H), 3.98 (m, 1H), 3.84(s, 3H), 3.66 (m, 1H), 3.51 (m, 2H), 3.17 (m, 1H), 2.81 (t, J=7.5 Hz,2H), 2.71 (s, 3H), 2.65 (s, 3H), 2.14 (s, 3H), 1.68-1.52 (m, 4H), 1.49(s, 9H), 1.39 (s, 9H); MS (FAB+) m/z 720.6 (M+H⁺) (FAB−) m/z 718.5(M−H⁺)

Synthesis of Structure (34)

Structure (34) was synthesized as follows. To a solution ofphenethylamide (structure (33), 25 mg, 0.035 mmol) in THF (5 ml) wasadded 18 mg of p-toluenesulfonic acid monohydrate (0.093 mmol). Thereaction mixture was stirred at rt overnight to give a baseline soot byTLC. The solution was concentrated in vacuo, and the residue washedtwice with ether removing excess pTsOH to give structure (34) as ayellowish-white solid, which was used without further purification. ¹HNMR (500 MHz, CDCl₃) was consistent with the expected product, however,individual peak assignment was difficult due to broadening. MS (ES+) m/z520.4 (M+H⁺).

Structure (34) was reacted with structure (9a) of Example 1 (in ananalogous manner to the procedure described in Example 2 for thesynthesis of structure (18)), followed by oxidation and deprotection (inan analogous manner as described with respect to the oxidation anddeprotection of structures (18) and (19), respectively) to providestructure (35) as identified in Table 9 below.

Example 11 Synthesis of Representative β-Sheet Mimetics

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (36)

Structure (36) was synthesized in an analogous fashion to compound (34)starting with benzylamine and structure (32). ¹H NMR (500 MHz, CDCl₃)was consistent with the expected product, however, individual peakassignment was difficult due to broadening. MS (FAB+) m/z 506.4 (M+H⁺).

Structure (36) was reacted with structure (9a) of Example 1 (in ananalogous manner to the procedure described in Example 2 for thesynthesis of structure (18)), followed by oxidation and deprotection (inan analogous manner as described with respect to the oxidation anddeprotection of structures (18) and (19), respectively) to providestructure (37) as identified in Table 9 below.

Example 12 Synthesis of Representative β-Sheet Mimetics

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (38)

Structure (38) was synthesized in an analogous fashion to structure (34)starting with p-chlorophenethylamine and structure (32). ¹H NMR (500MHz, CDCl₃) was consistent with the expected product, individual peakassignment was difficult due to broadening. MS (ES+) m/z 554.5 (M+H⁺).

Structure (38) was reacted with structure (9a) of Example 1 (in ananalogous manner to the procedure described in Example 2 for thesynthesis of structure (18)), followed by oxidation and deprotection (inan analogous manner as described with respect to the oxidation anddeprotection of structures (18) and (19), respectively) to providestructure (39) as identified in Table 9 below.

Example 13 Synthesis of Representative β-Sheet Mimetics

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (40)

Structure (40) was synthesized in an analogous fashion to compound (34)using p-methoxyphenethylamine and structure (32). ¹H NMR (500 MHz,CDCl₃) was consistent with the expected product, however, individualassignment was difficult due to broadening. MS (ES+) m/z 550.5 (M+H⁺).

Structure (40) was reacted with structure (9a) of Example 1 (in ananalogous manner to the procedure described in Example 2 for thesynthesis of structure (18)), followed by oxidation and deprotection (inan analogous manner as described with respect to the oxidation anddeprotection of structures (18) and (19), respectively) to providestructure (41) as identified in Table 9 below.

Example 14 Synthesis of Representative β-Sheet Mimetics

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (42)

Structure (42) was prepared as follows. In a 10 ml round-bottomed flaskwere added CH₂Cl₂ (10 ml), methyl 2,3-dimethylaminopropionatedihydrochloride (19.9 mg, 0.103 mmol, 1.5 eq), and diisopropylethylamine(53 ml, 0.304 mmol, 4.4 eq). This suspension was stirred magnetically atroom temperature for 1 h at which time was added the compound ofstructure (30) (50 mg, 0.068 mmol, 1 eq), mercury(II)chloride (82.4 mg,0.304 mmol, 4.4 eq), and mercury(II)oxide (25.7 mg, 0.120 mmol, 1.7 eq).The resulting yellow suspension was stirred for 16.5 h during which timethe suspension turned gray. The reaction was diluted with CH₂Cl₂ (50ml), washed with saturated aqueous NH₄Cl (5 ml), saturated aqueous NaCl(5 ml) and dried over Na₂SO₄. The cloudy suspension was filtered and thesolvent removed in vacuo. The white solid was purified on preparativethin-layer chromatography to produce the imidazoline structure (42)(25.3 mg, 52% yield) as a clear amorphous solid. R_(f) 0.11 (10%MeOH/CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 9.82 (s, 0.6H, N′H, mixture oftautomers), 9.78 (s, 0.4H, N″H), 8.35 (dd, J=4.3, 11 Hz, ¹H, N-5), 6.54(s, 1H, ArH), 5.08 (d, J=11 Hz, 1H, CHOH), 4.52 (m, 1H, imidazolineCH₂), 4.38 (d, J=21 Hz, 1H), 3.8-4.0 (m, 2H), 3.86 (s, 3H, CO₂CH ₃),3.767 (s, 3H, ArOCH₃), 3.5-3.7 (m, 2H, C-5 CH ₂), 3.16-3.27 (m, C-5 CH₂), 2.70 (s, 3H, ArCH₃), 2.63 (s, 3H, ArCH₃), 2.14 (s, 3H, ArCH₃),1.5-1.7 (m, 4H, C-3 and C-4 CH2), 1.49 (s, 9H, Boc), 1.46 (s, 9H, Boc);IR (film) 1725.56, 1685.68, 1618.36, 1585.45, 1207.09, 1148.85 cm⁻¹; MS(ES+) m/e 699.4 (M+H⁺).

Synthesis of Structure (43)

Structure (43) was synthesized as follows. In a 25 ml round-bottomedflask was placed the compound of structure (42) (230 mg, 0.33 mmol),CHCl₃ (5 ml) and MnO₂ (500 mg, 5.75 mmol, 17.4 eq) After stirring for 5h the suspension was filtered and the solid washed with methanol. Thesolvent was removed in vacuo and the residue was dissolved in ethylacetate (5 ml) and methanol (1 ml) and a fresh portion of MnO₂ (500 mg)was introduced and the reaction stirred for 15 h at room temperature.The solid was filtered and the solvent removed in vacuo. The residue waspurified via column chromatography on silica gel, eluting with 1:1 ethylacetate:hexane, then pure ethyl acetate, then 1:9 methanol:ethyl acetateto obtain the desired product (structure (43), 190 mg, 83% yield) as anamorphous solid.: R_(f) 0.64 (70:30-ethyl acetate:hexane); ¹H NMR (500MHz, CDCl₃) δ 10.70 (bs, 1H, imidazole NH), 9.70 (s, 1H), 8.28 (s, 1H),7.84 (s, 1H), 6.54 (s, 1H, ArH), 5.35 (m, 1H, aH), 5.25 (s, 1H, BocNH),3.926 (s, 3H), 3.840 (s, 3H), 3.15-3.40 (m, 2H), 2.682 (s, 3H), 2.133(s, 3H), 1.52-1.70 (m, 4H), 1.470 (s, 9H), 1.424 (s, 9H); IR (film)1724.68, 1619.03, 1277.72, 1151.93, 1120.61 cm⁻¹; MS (ES+) m/e 695.2(M+H⁺, 22), 717.2 (M+Na⁺, 100).

Synthesis of Structure (44)

Structure (44) was synthesized by the same method used to constructstructure (33) to structure (34). The product was used in the couplingwithout further purification.

Structure (44) was reacted with structure (9a) of Example 1 (in ananalogous manner to the procedure described in Example 2 for thesynthesis of structure (18)), followed by deprotection (in an analogousmanner as described with respect to the deprotection of structure (19)respectively) to provide structure (45) as identified in Table 9 below.In the preparation of structure (45), the coupling step was performedwith the carbonyl compound of structure (44), rather than with theanalogous hydroxy compound.

Example 15 Synthesis of Representative β-Sheet Mimetics

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (46)

Structure (46) was synthesized in an analogous fashion to structure (17)starting from structure (16) and thiazole. This compound was used in thecoupling step without further purification.

Structure (46) was reacted with structure (9a) of Example 1 (in ananalogous manner to the procedure described in Example 2 for thesynthesis of structure (18)), followed by oxidation and deprotection (inan analogous manner as described with respect to the oxidation anddeprotection of structures (13) and (19), respectively) to providestructure (47) as identified in Table 9 below.

Example 16 Synthesis of Representative β-Sheet Mimetics

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention.

Synthesis of Structure (48)

To a solution of α-Boc-β-Fmoc-2,3-diaminopropionic acid (818 mg, 1.92mmol) stirred in THF (5 ml) at −25° C. was added 4-methylmorpholine(0.23 ml, 2.1 mmol) followed by isobutylchloroformate (0.25 ml, 1.9mmol). The resulting suspension was stirred for 5 minutes and thenfiltered with the aid of 5 ml of THF. The filtrate was cooled in anice/water bath then sodium borohydride (152 mg, 0.40 mmol) dissolved inwater (2.5 ml) was added dropwise. The mixture was stirred for 15minutes then water (50 ml) was added and the mixture was extracted withCH₂Cl₂ (3×50 ml). The combined extracts were washed with brine, driedover anhydrous sodium sulfate and filtered. Concentration of thefiltrate under vacuum yielded a pale yellow solid that was purified byflash chromatography (50% ethyl acetate/hexanes eluent) to give 596 mgof the alcohol as a white solid.

The alcohol (224 mg, 0.543 mmol) was dissolved in methylene chloride andDess-Martin periodinane (262 mg, 0.64 mmol) was added. The mixture wasstirred at room temperature for 1 h then diluted with ethyl acetate (50ml) and extracted sequentially with 10% aqueous Na₂S₂O₃, saturatedaqueous NaHCO₃, and brine. The organic solution was dried over anhydroussodium sulfate, filtered and concentrated under vacuum to a white solid.Purification of the solid by flash chromatography yielded 169 mg of thealdehyde structure (48) as a white solid.

Synthesis of Structure (49)

Structure (49) was synthesized in an analogous fashion to structure (17)starting from structure (48) and benzothiazole. This compound was usedas a 1:1 mixture of diastereomers in the coupling step (described below)without further purification. MS (EI+): m/z 446.4 (M+H⁺).

Synthesis of Structure (50)

Structure (49) and bicyclic acid structure (9a) (27 mg, 0.069 mmol) andHOBt hydrate (71 mg, 0.46 mmol) were dissolved in THF (1 ml) anddiisopropylethylamine (0.0.059 ml, 0.34 mmol) was added followed by EDC(19 mg, 0.099 mmol). The mixture was stirred at room temperature for 20h then diluted with ethyl acetate and extracted sequentially with 5%aqueous citric acid, saturated aqueous sodium bicarbonate, water andbrine. The organic solution was dried over anhydrous sodium sulfate,filtered and concentrated under vacuum to 61 mg of a yellow foam. ¹H NMRanalysis indicated a mixture of diastereomeric amides.

The foam was dissolved in CH₃CN and diethylamine was added. The solutionwas stirred at room temperature for 30 minutes then concentrated undervacuum to a yellow foam. The foam was rinsed with hexanes and dissolvedin DMF (0.5 ml). In a separate flask, carbonyldiimidazole (16 mg, 0.99mmol) and guanidine hydrochloride (10 mg, 0.10 mmol) were dissolved inDMF (1 ml) and diisopropylethylamine (0.035 ml, 0.20 mmol) was addedfollowed by DMAP (1 mg). The solution was stirred for 1.5 h at roomtemperature then the solution of amine was added and stirring wascontinued for 16 h. The solution was concentrated under vacuum thenwater was added to the residue and the mixture was extracted with ethylacetate (3×25 ml). The combined extracts were washed with brine, driedover anhydrous sodium sulfate and filtered. Concentration of thefiltrate under vacuum yielded 58 mg of structure (50) as a yellow foam.MS (ES+): m/z 680.6 (M+H⁺).

Structure (50) was oxidized to provide the corresponding ketone ofstructure (51).

Example 17 Activities of Representative β-Sheet Mimetics as a ProteaseInhibitor

This example illustrates the ability of further representative β-sheetmimetics of this invention to function as an inhibitor for thrombin,Factor VII, Factor X, Factor XI, tryptase, aPC, plasmin, tPA, urokinasethrombin thrombomodulin complex and trypsin. The β-sheet mimetics of thestructures listed in Table 9 had the inhibition activities shown inTable 10.

The proteinase inhibitor assays were performed as described in Example9. The assay for thrombin-thrombomodulin complex was conducted as forthrombin except that prior to the addition of inhibitor and substrate,thrombin was preincubated with 4 nM thrombomodulin for 20 minutes atroom temperature.

TABLE 9 Structures, Synthetic Precursors, and Physical Data for VariousSerine Protease Inhibitors

Struc- ture Number B^(δ) R₄ R₅

M.S. (ES+) HPLC* R.T. (min) (47) N

(46) 513.5 (M + H⁺) 15.9 (20b) N

(17) 563.5 (M + H⁺) 17.9 (37) N

(36) 563.6 (M + H⁺) 16.9 (39) N

(38) 611.3 (M + H⁺) 19.8 (29a)^(ε) CH

(17) 562.4 (M + H⁺) 21.2 (35) N

(34) 577.4 (M + H⁺) 18.1 (45) N

(44) 554.2 (M + H⁺) 15.7 (51) N

(49) 578.3 (M + H⁺) 22.3 (29b) CH

(17) FAB 562.4 (M + H⁺) 21.5 (41) N

(40) 607.4 (M + H⁺) 18.2 (13) N

Arg(Mtr) —CH₂Cl 477.9 (M + H⁺) 14.9 ^(δ)The stereochemistry of thetemplate for B = CH is (3R, 6R, 9S) except where noted (see footnote ε).^(ε)Template stereochemistry is (3S, 6R, 9S). *HPLC was performed on areverse phase C-18 column using a gradient of 0-90% acetonitrile/water,0.1% TFA.

TABLE 10 Ki (M) Inhihition Activity of Various Compounds Against SerineProteases Structure Factor Factor Number Thrombin VII Factor X XIUrokinase T.T.C.^(a) aPC^(b) Plasmin tPA^(c) Trypsin Tryptase 357.10E-11 1.64E-08 3.45E-07^(d) 2.70E-11 37 7.32E-11 7.73E-11 29b8.50E-11 2.00E-07 1.93E-08 7.53E-08 3.35E-07 8.80E 11 1.25E-06 2.51E-079.29E-08 2.30E-10 9.00E-09 39 3.10E-10 41 4.50E-10 20b 6.50E-10 2.70E-072.22E-07 2.70E-08 6.00E-07 3.32E-06 4.15E-07 4.95E-07 6.20E-10 1.24E-0847 2.40E-09 9.68E-07 1.50E-06^(e) 1.90E-09 45 5.40E-09 2.96E-05 3.80E-051.24E-06 6.90E-09 2.56E-05 2.38E-05 1.72E-05 5.24E-08 1.65E-06 517.25E-09 4.26E-06 5.70E-05 1.73E-06 3.79E-08 29d 1.04E-08 1.77E-064.65E-06^(d) 3.91E-07 5.40E-10 13d 1.20E-09 1.40E-07 3.86E-07^(e)9.27E-07 5.28E-07 9.78E-07 6.32E-07 1.60E-07 ^(a)Thrombin thrombomodulincomplex, ^(b)activated Protein C, ^(c)tissue Plasminogen Activator,^(d)IC50, ^(e)bovine plasma

Example 18 Effect of Representative β-Sheet Mimetics on PlateletDeposition in a Vascular Graft

The effect of compounds of the invention on platelet deposition in avascular graft was measured according to the procedure of Hanson et al.“Interruption of acute platelet-dependent thrombosis by syntheticantithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethylketone” Proc.Natl. Acad. Sci., USA 85:3148-3188, (1988), except that the compound wasintroduced proximal to the shunt as described in Kelly et al., Proc.Natl. Acad. Sci., USA 89:6040-6044 (1992). The results are shown inFIGS. 1, 2 and 3 for structures (20b), (39) and (29b), respectively.

Example 19 Synthesis of Representative β-Sheet Mimetics

This example illustrates the synthesis of a further representativeβ-sheet mimetic of this invention having the structure shown below.

Structure (52) may be synthesized employing the following intermediate(53) in place or intermediate (16) in Example 2:

Intermediate (53) may be synthesized by the following reaction scheme:

Alternatively, intermediate (53) may be synthesized by the followingreaction scheme:

From the foregoing, it will be appreciated that, although specificembodiments of this invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except by the appended claims.

What is claimed is:
 1. A bicyclic ring compound having the structure:

wherein R₁, R₂ and R₃ are independently selected from amino acid sidechain moieties and derivatives thereof; A is —C(═O)—, —(CH₂)₁₋₄—,—C(═O)(CH₂)₁₋₃—, —(CH₂)₁₋₂O— or —(CH₂)₁₋₂S—; B is CH; C is —C(═O)—,—(CH₂)₁₋₃—, —O—, —S—, —O—(CH₂)₁₋₂— or —S(CH₂)₁₋₂—; Y and Z represent theremainder of the molecule; and any two adjacent CH groups of thebicyclic ring may form a double bond; with the provisos that (i) R₁ isnot hydrogen, (ii) when R₁ is benzyl, R₂ and R₃ are both hydrogen, and Ais —CH₂CH₂—, then C is not —CH₂—, (iii) when R₁ is methyl, R₂ and R₃ areboth hydrogen, and A is —CH₂O—, then C is not —CH₂—, and (iv) when R₁ isbenzyl, R₂ and R₃ are both hydrogen, and A is —CH₂—, then C is not —S—.2. The compound of claim 1 having the structure:

wherein A is —C(═O)—, —(CH₂)₁₋₄— or —C(═O)(CH₂)₁₋₃—; C is —C(═O)— or—CH₂)₁₋₃, and the bicyclic ring is saturated.
 3. The compound of claim 2having the structure:

wherein n is an integer from to 4 and p is an integer from 1 to
 3. 4.The compound of claim 2 having the structure:

wherein p is an integer from 1 to
 3. 5. The compound of claim 2 havingthe structure:

wherein n is an integer from 1 to
 4. 6. The compound of claim 1 havingthe structure:

wherein A is —(CH₂)₁₋₄—, —(CH₂)₁₋₂O— or —(CH₂)₁₋₂S—; C is —(CH₂)₁₋₃—,—O—, —S—, —O(CH₂)₁₋₂— or —S(CH₂)₁₋₂—, and the bicyclic ring issaturated.
 7. The compound of claim 6 having the structure:

wherein n is an integer from 1 to
 4. 8. The compound of claim 6 havingthe structure:

wherein m is an integer from 1 to 2; and p is an integer from 1 to
 3. 9.The compound of claim 6 having the structure:

wherein m is an integer from 1 to 2; and p is an integer from 1 to 3.10. The compound of claim 6 having the structure:

wherein p is an integer from 1 to
 3. 11. The compound of claim 6 havingthe structure:

wherein p is an integer from 1 to
 3. 12. The compound of claim 6 havingthe structure:

wherein p is an integer from 1 to 3; and m is an integer from 1 to 2.13. The compound of claim 6 having the structure:

wherein p is an integer from 1 to 3; and m is an integer from 1 to 2.14. The compound of claim 6 having the structure:

wherein p is an integer from 1 to
 3. 15. The compound of claim 14wherein A is —CH₂CH₂—, p is 1 and R₂ and R₃ are both hydrogen.